Oligonucleotide compositions and methods thereof

ABSTRACT

Among other things, the present disclosure relates to chirally controlled oligonucleotides of select designs, chirally controlled oligonucleotide compositions, and methods of making and using the same. In some embodiments, a provided chirally controlled oligonucleotide composition provides different cleavage patterns of a nucleic acid polymer than a reference oligonucleotide composition. In some embodiments, a provided chirally controlled oligonucleotide composition provides single site cleavage within a complementary sequence of a nucleic acid polymer. In some embodiments, a chirally controlled oligonucleotide composition has any sequence of bases, and/or pattern or base modifications, sugar modifications, backbone modifications and/or stereochemistry, or combination of these elements, described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/746,220, filed Jan. 19, 2018, issued as U.S. Pat. No. 10,479,995,which is a U.S. National Stage Application of International PCTApplication Number PCT/US2016/043542, filed Jul. 22, 2016, which claimspriority to United States Provisional Application Nos. 62/195,779, filedJul. 22, 2015, 62/236,847, filed Oct. 2, 2015, and 62/331,960, filed May4, 2016, the entirety of each of which is incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 25, 2019, isnamed SL.txt and is 420,949 bytes in size.

BACKGROUND

Oligonucleotides are useful in therapeutic, diagnostic, research andnanomaterials applications. The use of naturally occurring nucleic acids(e.g., unmodified DNA or RNA) for therapeutics can be limited, forexample, because of their instability against extra- and intracellularnucleases and/or their poor cell penetration and distribution. There isa need for new and improved oligonucleotides and oligonucleotidecompositions, such as, e.g., new antisense and siRNA oligonucleotidesand oligonucleotide compositions.

SUMMARY

Among other things, the present disclosure encompasses the recognitionthat structural elements of oligonucleotides, such as base sequence,chemical modifications (e.g., modifications of sugar, base, and/orinternucleotidic linkages, and patterns thereof), and/or stereochemistry(e.g., stereochemistry of backbone chiral centers (chiralinternucleotidic linkages), and/or patterns thereof), can havesignificant impact on properties, e.g., activities, of oligonucleotides.In some embodiments, the present disclosure demonstrates thatoligonucleotide compositions comprising oligonucleotides with controlledstructural elements, e.g., controlled chemical modification and/orcontrolled backbone stereochemistry patterns, provide unexpectedproperties, including but not limited to those described herein. In someembodiments, the present disclosure demonstrates that combinations ofchemical modifications and stereochemistry can provide unexpected,greatly improved properties (e.g., bioactivity, selectivity, etc.). Insome embodiments, the present disclosure provides an oligonucleotidecomposition having a particular sequence of bases, and/or pattern ofsugar modifications (e.g., 2′-OMe, 2′-F, 2′-MOE, etc.), and/or patternor base modifications (e.g., 5-methylcytosine), and/or pattern ofbackbone modifications (phosphate or phosphorothioate), and/or patternof stereochemistry of backbone modifications (e.g., eachphosphorothioate is Sp or Rp).

In some embodiments, modifications of internucleotidic linkages canconvert phosphorus atoms in modified linkages into chiral centers. Forexample, in a phosphorothioate (PS) modification, one of thenon-bridging oxygen (O) atoms bonded to a phosphorus (P) atom isreplaced with a sulfur (S) atom. A consequence of using PS modificationin oligonucleotide synthesis is that it creates a chiral center atphosphorus, which can have either an “Sp” or “Rp” configuration. Forinstance, a conventional stereorandom PS-modified oligonucleotidecomposition having 19 PS linkages [e.g., having 20 nucleotides inlength, 19 PS modifications, each with two possible stereochemistries(Sp or Rp) at each PS modification] is a random mixture of over 500,000(2¹⁹) stereoisomers, each having the same nucleotide sequence (e.g.,sequence of bases) but differing in the stereochemistry along theirbackbones; such a composition is a “stereorandom” oligonucleotidecomposition. In some embodiments, in contrast to stereorandomcompositions, a chirally controlled oligonucleotide composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have acommon base sequence and length, a common pattern of backbone linkages,and a common pattern of backbone chiral centers. In some embodiments,some oligonucleotide compositions are stereopure (i.e., a chirallycontrolled oligonucleotide composition), wherein the stereochemistry ateach PS is defined (Sp or Rp). In some embodiments, in a stereorandomcompositions of oligonucleotides, the various oligonucleotides can havethe same base sequence, same pattern of sugar modifications (e.g.,2′-OMe, 2′-F, 2′-OME, etc.), same pattern of base modifications (e.g.,5-methylcytosine), and same pattern of backbone modifications (phosphateor PS), but different patterns of backbone chiral centers, and theirlevels are random from non-stereocontrolled synthesis (notpre-determined as through stereocontrolled synthesis as certain methodsexemplified herein using chiral auxilier). A chirally controlledoligonucleotide composition can be selected to have greater desiredbiological activity (e.g., greater activities, efficiency in RNAinterference or RNAse H-mediated pathways, etc.) and decreased undesiredactivity (e.g., undesired immunogenicity, toxicity, etc.) than astereorandom preparation of oligonucleotides of the same base sequence.In some embodiments, a chirally controlled oligonucleotide compositionis better able to differentiate between a mutant (mu) and a wild-type(wt) HTT sequence (with a single nt difference).

Among other things, the present disclosure encompasses the recognitionthat stereorandom oligonucleotide preparations contain a plurality ofdistinct chemical entities that differ from one another, e.g., in thestereochemical structure of individual backbone chiral centers withinthe oligonucleotide chain. Without control of stereochemistry ofbackbone chiral centers, stereorandom oligonucleotide preparationsprovide uncontrolled compositions comprising undetermined levels ofoligonucleotide stereoisomers. Even though these stereoisomers may havethe same base sequence, they are different chemical entities at leastdue to their different backbone stereochemistry, and they can have, asdemonstrated herein, different properties, e.g., bioactivities. Amongother things, the present disclosure provides new compositions that areor contain particular stereoisomers of oligonucleotides of interest. Insome embodiments, a particular stereoisomer may be defined, for example,by its base sequence, its length, its pattern of backbone linkages, andits pattern of backbone chiral centers. As is understood in the art, insome embodiments, base sequence may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotideand/or to the hybridization character (i.e., the ability to hybridizewith particular complementary residues) of such residues.

The present disclosure demonstrates, among other things, that individualstereoisomers of a particular oligonucleotide can show differentstability and/or activity (e.g., functional and/or toxicity properties)from each other. Moreover, the present disclosure demonstrates thatstability and/or activity improvements achieved through inclusion and/orlocation of particular chiral structures within an oligonucleotide canbe comparable to, or even better than those achieved through use ofparticular backbone linkages, residue modifications, etc. (e.g., throughuse of certain types of modified phosphates [e.g., phosphorothioate,substituted phosphorothioate, etc.], sugar modifications [e.g.,2′-modifications, etc.], and/or base modifications [e.g., methylation,etc.]).

Among other things, the present disclosure recognizes that, in someembodiments, properties (e.g., stability and/or activities) of anoligonucleotide can be adjusted by optimizing its pattern of backbonechiral centers, optionally in combination with adjustment/optimizationof one or more other features (e.g., linkage pattern, nucleosidemodification pattern, etc.) of the oligonucleotide.

In some embodiments, the present disclosure provides compositions ofoligonucleotides, wherein the oligonucleotides have a common pattern ofbackbone chiral centers which, unexpectedly, greatly enhances thestability and/or biological activity of the oligonucleotides. In someembodiments, a pattern of backbone chiral centers provides increasedstability. In some embodiments, a pattern of backbone chiral centersprovides surprisingly increased activity. In some embodiments, a patternof backbone chiral centers provides increased stability and activity. Insome embodiments, when an oligonucleotide is utilized to cleave anucleic acid polymer, a pattern of backbone chiral centers, surprisinglyby itself, changes the cleavage pattern of a target nucleic acidpolymer. In some embodiments, a pattern of backbone chiral centerseffectively prevents cleavage at secondary sites. In some embodiments, apattern of backbone chiral centers creates new cleavage sites. In someembodiments, a pattern of backbone chiral centers minimizes the numberof cleavage sites. In some embodiments, a pattern of backbone chiralcenters minimizes the number of cleavage sites so that a target nucleicacid polymer is cleaved at only one site within the sequence of thetarget nucleic acid polymer that is complementary to theoligonucleotide. In some embodiments, a pattern of backbone chiralcenters enhances cleavage efficiency at a cleavage site. In someembodiments, a pattern of backbone chiral centers of the oligonucleotideimproves cleavage of a target nucleic acid polymer. In some embodiments,a pattern of backbone chiral centers increases selectivity. In someembodiments, a pattern of backbone chiral centers minimizes off-targeteffect. In some embodiments, a pattern of backbone chiral centersincrease selectivity, e.g., cleavage selectivity between two targetsequences differing only by a single nucleotide polymorphism (SNP). Insome embodiments, a pattern of backbone chiral centers comprises,comprises one or more repeats of, or is (Sp)_(m)(Rp)_(n),(Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m).In some embodiments described herein, m is 1-50; and n is 1-10; and t is1-50. In some embodiments, a pattern of backbone chiral centerscomprises or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m. In some embodiments, a pattern of backbone chiralcenters comprises or is (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein m>2. In some embodiments, a pattern ofbackbone chiral centers is a sequence comprising at least 5, 6, 7, 8, 9,or 10 or more consecutive (Sp) positions. In some embodiments, a patternof backbone chiral centers is a sequence comprising at least 5consecutive (Sp) positions. In some embodiments, a pattern of backbonechiral centers is a sequence comprising at least 8 consecutive (Sp)positions. In some embodiments, a pattern of backbone chiral centers isa sequence comprising at least 10 consecutive (Sp) positions. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp). In some embodiments, apattern of backbone chiral centers is a sequence consisting of all (Sp)with a single (Rp) at or adjacent to the position of a SNP. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format. In some embodiments, a pattern of backbone chiralcenters is a sequence consisting of all (Sp) with a single (Rp), whereinthe molecule has a wing-core-wing format, wherein the wing on the 5′ endis 1-9 nt long, the core is 1-15 nt long, and the wing on the 3′ end is1-9 nt long. In some embodiments, a pattern of backbone chiral centersis a sequence consisting of all (Sp) with a single (Rp), wherein themolecule has a wing-core-wing format, wherein the wing on the 5′ end is5 nt long, the core is 1-15 nt long, and the wing on the 3′ end is 5 ntlong. In some embodiments, a pattern of backbone chiral centers is asequence consisting of all (Sp) with a single (Rp), wherein the moleculehas a wing-core-wing format, wherein the wing on the 5′ end is 1-9 ntlong, the core is 10 nt long, and the wing on the 3′ end is 1-9 nt long.In some embodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long, and atleast one wing comprises a nucleotide with a 2′-OMe modification. Insome embodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein each wing comprises at least onenucleotide with a 2′-OMe modification. In some embodiments, a pattern ofbackbone chiral centers is a sequence consisting of all (Sp) with asingle (Rp), wherein the molecule has a wing-core-wing format, whereineach nucleotide in both wings has a 2′-OMe modification. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long, and eachnucleotide in each wing has a 2′-OMe modification. In some embodiments,the oligonucleotide is single-stranded and has a wing-core-wing format,wherein the wing on the 5′ end of the molecule comprises 4 to 8 nt, eachof which has a 2′-OMe modification and wherein the nt at the 5′ end ofthe molecule has a phosphorothioate in the Sp conformation; the corecomprises 8 to 12 nt, each of which is DNA (2′-H), wherein each has aphosphorothioate in the Sp position except one nt which has thephosphorothioate in the Rp position; and wherein the wing on the 3′ endof the molecule comprises 4 to 8 nt, each of which has a 2′-OMemodification, and wherein the nt at the 3′ end of the molecule comprisesa phosphorothioate in the Sp conformation. In some embodiments, theoligonucleotide is single-stranded and has a wing-core-wing format,wherein the wing on the 5′ end of the molecule comprises 6 nt, each ofwhich has a 2′-OMe modification and wherein the nt at the 5′ end of themolecule has a phosphorothioate in the Sp conformation; the corecomprises 10 nt, each of which is DNA (2′-H), wherein each has aphosphorothioate in the Sp position except one nt which has thephosphorothioate in the Rp position; and wherein the wing on the 3′ endof the molecule comprises 6 nt, each of which has a 2′-OMe modification,and wherein the nt at the 3′ end of the molecule comprises aphosphorothioate in the Sp conformation.

In some embodiments, the present disclosure recognizes that chemicalmodifications, such as modifications of nucleosides and internucleotidiclinkages, can provide enhanced properties. In some embodiments, thepresent disclosure demonstrates that combinations of chemicalmodifications and stereochemistry can provide unexpected, greatlyimproved properties (e.g., bioactivity, selectivity, etc.). In someembodiments, chemical combinations, such as modifications of sugars,bases, and/or internucleotidic linkages, are combined withstereochemistry patterns, e.g., (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m), to provideoligonucleotides and compositions thereof with surprisingly enhancedproperties. In some embodiments, a provided oligonucleotide compositionis chirally controlled, and comprises a combination of 2′-modificationof one or more sugar moieties, one or more natural phosphate linkages,one or more phosphorothioate linkages, and a stereochemistry pattern of(Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m),wherein m>2.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotidesdefined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that atleast about 10% of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

Among other things, the present disclosure recognizes that combinationsof oligonucleotide structural elements (e.g., patterns of chemicalmodifications, backbone linkages, backbone chiral centers, and/orbackbone phosphorus modifications) can provide surprisingly improvedproperties such as bioactivities. In some embodiments, the presentdisclosure provides an oligonucleotide composition comprising apredetermined level of oligonucleotides which comprise one or more wingregions and a common core region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

In some embodiments, in an oligonucleotide comprising a wing-core-wingformat, a “wing” is a portion of the oligonucleotide on the 5′ or 3′ endof the core, with the “core” (alternatively designated a “gap”) betweenthe two wings. In some embodiments, an oligonucleotide can have a singlewing and a single core; in such cases, the wing is on the 5′ or the 3′end of the oligonucleotide. A wing and core can be defined by any ofseveral structural elements (e.g., modifications or patterns ofmodifications of sugar, base, backbone or backbone stereochemistry,etc.). In some embodiments, a wing and core is defined by nucleosidemodifications, wherein a wing comprises a nucleoside modification thatthe core region does not have. In some embodiments, oligonucleotides inprovided compositions have a wing-core structure of nucleosidemodification. In some embodiments, oligonucleotides in providedcompositions have a core-wing structure of nucleoside modification. Insome embodiments, oligonucleotides in provided compositions have awing-core-wing structure of nucleoside modification. In someembodiments, a wing and core is defined by modifications of the sugarmoieties. In some embodiments, a wing and core is defined bymodifications of the base moieties. In some embodiments, each sugarmoiety in the wing region has the same 2′-modification which is notfound in the core region. In some embodiments, each sugar moiety in thewing region has the same 2′-modification which is different than anysugar modifications in the core region. In some embodiments, each sugarmoiety in the wing region has the same 2′-modification, and the coreregion has no 2′-modifications. In some embodiments, when two or morewings are present, each sugar moiety in a wing region has the same2′-modification, yet the common 2′-modification in a first wing regioncan either be the same as or different from the common 2′-modificationin a second wing region.

In some embodiments, each wing comprises at least one chiralinternucleotidic linkage and at least one natural phosphate linkage. Insome embodiments, each wing comprises at least one modified sugarmoiety. In some embodiments, each wing sugar moiety is modified. In someembodiments, a wing sugar moiety is modified by a modification that isabsent from the core region. In some embodiments, a wing region only hasmodified internucleotidic linkages at one or both of its ends. In someembodiments, a wing region only has a modified internucleotidic linkageat its 5′-end. In some embodiments, a wing region only has a modifiedinternucleotidic linkage at its 3′-end. In some embodiments, a wingregion only has modified internucleotidic linkages at its 5′- and3′-ends. In some embodiments, a wing is to the 5′-end of a core, and thewing only has a modified internucleotidic linkage at its 5′-end. In someembodiments, a wing is to the 5′-end of a core, and the wing only has amodified internucleotidic linkage at its 3′-end. In some embodiments, awing is to the 5′-end of a core, and the wing only has modifiedinternucleotidic linkages at both its 5′- and 3′-ends. In someembodiments, a wing is to the 3′-end of a core, and the wing only has amodified internucleotidic linkage at its 5′-end. In some embodiments, awing is to the 3′-end of a core, and the wing only has a modifiedinternucleotidic linkage at its 3′-end. In some embodiments, a wing isto the 3′-end of a core, and the wing only has modified internucleotidiclinkages at both its 5′- and 3′-ends. In some embodiments, themodification(s) to the sugar moiety or internucleotidic linkage or othermodifications in one wing can differ from those in another wing.

In some embodiments, each internucleotidic linkage within a core regionis modified. In some embodiments, each internucleotidic linkage within acore region is chiral. In some embodiments, a core region has a patternof backbone chiral centers of (Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m). In someembodiments, a core region has a pattern of backbone chiral centers of(Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m),wherein m>2. Among other things, the present disclosure demonstratesthat, in some embodiments, such patterns can provide or enhancecontrolled cleavage of a target sequence, e.g., an RNA sequence.

In some embodiments, oligonucleotides in provided compositions have acommon pattern of backbone phosphorus modifications. In someembodiments, a provided composition is an oligonucleotide compositionthat is chirally controlled in that the composition contains apredetermined level of oligonucleotides of an individual oligonucleotidetype, wherein an oligonucleotide type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

As noted above and understood in the art, in some embodiments, the basesequence of an oligonucleotide may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in theoligonucleotide and/or to the hybridization character (i.e., the abilityto hybridize with particular complementary residues) of such residues.

In some embodiments, a particular oligonucleotide type may be defined by

1A) base identity;

1B) pattern of base modification;

1C) pattern of sugar modification;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

Thus, in some embodiments, oligonucleotides of a particular type mayshare identical bases but differ in their pattern of base modificationsand/or sugar modifications. In some embodiments, oligonucleotides of aparticular type may share identical bases and pattern of basemodifications (including, e.g., absence of base modification), butdiffer in pattern of sugar modifications.

In some embodiments, oligonucleotides of a particular type arechemically identical in that they have the same base sequence (includinglength), the same pattern of chemical modifications to sugar and basemoieties, the same pattern of backbone linkages (e.g., pattern ofnatural phosphate linkages, phosphorothioate linkages, phosphorothioatetriester linkages, and combinations thereof), the same pattern ofbackbone chiral centers (e.g., pattern of stereochemistry (Rp/Sp) ofchiral internucleotidic linkages), and the same pattern of backbonephosphorus modifications (e.g., pattern of modifications on theinternucleotidic phosphorus atom, such as —S⁻, and -L-R¹ of formula I).

In some embodiments, the sequence of the oligonucleotide comprises orconsists of the sequence of any oligonucleotide disclosed herein. Insome embodiments, the sequence of the oligonucleotide comprises orconsists of the sequence of any oligonucleotide selected from Tables N1,N2, N3, N4 and 8. In some embodiments, the sequence of theoligonucleotide comprises or consists of the sequence of anyoligonucleotide selected from Tables N1A, N2A, N3A, N4A and 8. In someembodiments, the sequence of the oligonucleotide in a stereopure(chirally controlled) oligonucleotide composition comprises or consistsof the sequence of WV-1092, WVE120101, WV-2603 or WV-2595. In someembodiments, a sequence of an oligonucleotide includes any one or moreof: base sequence (including length); pattern of chemical modificationsto sugar and base moieties; pattern of backbone linkages; pattern ofnatural phosphate linkages, phosphorothioate linkages, phosphorothioatetriester linkages, and combinations thereof; pattern of backbone chiralcenters; pattern of stereochemistry (Rp/Sp) of chiral internucleotidiclinkages; pattern of backbone phosphorus modifications; pattern ofmodifications on the internucleotidic phosphorus atom, such as —S⁻, and-L-R¹ of formula I.

Among other things, the present disclosure recognizes the challenge ofstereoselective (rather than stereorandom or racemic) preparation ofoligonucleotides. Among other things, the present disclosure providesmethods and reagents for stereoselective preparation of oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10)internucleotidic linkages, and particularly for oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiralinternucleotidic linkages. In some embodiments, in a stereorandom orracemic preparation of oligonucleotides, at least one chiralinternucleotidic linkage is formed with less than 90:10, 95:5, 96:4,97:3, or 98:2 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 90:10,95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, fora stereoselective or chirally controlled preparation ofoligonucleotides, each chiral internucleotidic linkage is formed withgreater than 95:5 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 96:4diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 97:3diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 98:2diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 99:1diastereoselectivity. In some embodiments, diastereoselectivity of achiral internucleotidic linkage in an oligonucleotide may be measuredthrough a model reaction, e.g. formation of a dimer under essentiallythe same or comparable conditions wherein the dimer has the sameinternucleotidic linkage as the chiral internucleotidic linkage, the5′-nucleoside of the dimer is the same as the nucleoside to the 5′-endof the chiral internucleotidic linkage, and the 3′-nucleoside of thedimer is the same as the nucleoside to the 3′-end of the chiralinternucleotidic linkage.

Among other things, it is surprisingly found that certain providedoligonucleotide compositions achieve unprecedented control of cleavageof target sequences, e.g., cleavage of target RNA by RNase H. In someembodiments, the present disclosure demonstrates that precise control ofchemical and stereochemical attributes of oligonucleotides achievesimproved activity of oligonucleotide preparations as compared withotherwise comparable preparations for which stereochemical attributesare not controlled. Among other things, the present disclosurespecifically demonstrates improved rate, degree, and or specificity ofcleavage of nucleic acid targets to which provided oligonucleotideshybridize.

In some embodiments, the present disclosure provides various uses ofoligonucleotide compositions. Among other things, the present disclosuredemonstrates that by controlling structural elements ofoligonucleotides, such as base sequence, chemical modifications,stereochemistry, etc., properties of oligonucleotides can be greatlyimproved. For example, in some embodiments, the present disclosureprovides methods for highly selective suppression of transcripts of atarget nucleic acid sequence. In some embodiments, the presentdisclosure provides methods for treating a subject by suppressingtranscripts from a disease-causing copy (e.g., a disease-causingallele). In some embodiments, the present disclosure provides methodsfor designing and preparing oligonucleotide compositions withsurprisingly enhanced activity and/or selectivity when suppressing atranscript of a target sequence. In some embodiments, the presentdisclosure provides methods for designing and/or preparingoligonucleotide compositions which provide allele-specific suppressionof a transcript from a target nucleic acid sequence.

In some embodiments, the present disclosure provides a method forcontrolled cleavage of a nucleic acid polymer, the method comprisingsteps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type.

In some embodiments, the present disclosure provides a method foraltering a cleavage pattern observed when a nucleic acid polymer whosenucleotide sequence includes a target sequence is contacted with areference oligonucleotide composition that comprises oligonucleotideshaving a particular base sequence and length, which particular basesequence is or comprises a sequence that is complementary to the targetsequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

1) the particular base sequence and length;

2) a particular pattern of backbone linkages; and

3) a particular pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a method forsuppression of a transcript from a target nucleic acid sequence forwhich one or more similar nucleic acid sequences exist within apopulation, each of the target and similar sequences contains a specificnucleotide characteristic sequence element that defines the targetsequence relative to the similar sequences, the method comprising stepsof:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

In some embodiments, the present disclosure provides a method forsuppression of a transcript from a target nucleic acid sequence forwhich one or more similar nucleic acid sequences exist within apopulation, each of the target and similar sequences contains a specificnucleotide characteristic sequence element that defines the targetsequence relative to the similar sequences, the method comprising stepsof:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

In some embodiments, transcripts of the target nucleic acid sequence aresuppressed at a greater level than a level of suppression observed forany one of the similar nucleic acid sequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides of a particularoligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, a nucleotide characteristic sequence comprises amutation that defines the target sequence relative to other similarsequences. In some embodiments, a nucleotide characteristic sequencecomprises a point mutation that defines the target sequence relative toother similar sequences. In some embodiments, a nucleotidecharacteristic sequence comprises a SNP that defines the target sequencerelative to other similar sequences.

In some embodiments, the present disclosure provides a method forpreparing an oligonucleotide composition comprising oligonucleotides ofa particular sequence, which composition provides selective suppressionof a transcript of a target sequence, comprising providing a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence which is the same as the particular sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises(Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein:

m is 1-50;

n is 1-10;

t is 1-50; and

each Np is independent Rp or Sp.

In general, activities of oligonucleotide compositions as describedherein can be assessed using any appropriate assay. Relative activitiesfor different compositions (e.g., stereocontrolled vsnon-stereocontrolled, and/or different stereocontrolled compositions)are typically desirably determined in the same assay, in someembodiments substantially simultaneously and in some embodiments withreference to historical results.

Those of skill in the art will be aware of and/or will readily be ableto develop appropriate assays for particular oligonucleotidecompositions. The present disclosure provides descriptions of certainparticular assays, for example that may be useful in assessing one ormore features of oligonucleotide composition behavior with respect toRNAse H cleavage of a target sequence.

For example, certain assays that may be useful in the assessment of oneor more features (e.g., rate, extent, and/or selectivity of cleavage) ofRNase H cleavage may include an assay as described in any assaydescribed and/or exemplified herein (e.g., in one or more of Examples 4,9-10, 12, 14, 17-20, etc.).

In some embodiments, the present disclosure recognizes that a basesequence can impact properties of oligonucleotides. The presentdisclosure demonstrates that chemical and stereochemical modifications,combined with designed base sequences, can provide oligonucleotidecompositions with unexpectedly improved properties (e.g., surprisinglyhigher activity, and/or selectivity, etc.). In some embodiments,oligonucleotides having a common base sequence complementary to acharacteristic sequence element of a target nucleic acid sequenceprovide better activity compared to another common base sequencecomplementary to the characteristic sequence element of a target nucleicacid sequence. In some embodiments, oligonucleotides having a commonbase sequence complementary to a characteristic sequence element of atarget nucleic acid sequence provide better selectivity compared toanother common base sequence complementary to the characteristicsequence element of a target nucleic acid sequence.

In some embodiments, a composition of oligonucleotides having a commonbase sequence complementary to a characteristic sequence element of atarget nucleic acid sequence, when compared to another composition ofoligonucleotides having another common base sequence complementary tothe characteristic sequence element of the target nucleic acid sequence,provides higher cleavage rate of a transcript from the target nucleicacid sequence, and/or a cleavage pattern which has only one majorcleavage site, and the major cleavage site is within or close to thenucleotide characteristic sequence. In some embodiments, a compositionof oligonucleotides having a complementary common base sequence, whencompared to another composition of oligonucleotides having anothercomplementary common base sequence, provide higher cleavage rate of atranscript from the target nucleic acid sequence, and a cleavage patternwhich has only one major cleavage site, and the major cleavage site iswithin or close to a nucleotide characteristic sequence. In someembodiments, greater than 50%, 60%, 70%, 80% or 90% of cleavage occursat the one major cleavage site, for example, when measured by a suitablemethod, e.g., an RNase H assay. In some embodiments, a composition ofoligonucleotides having a complementary common base sequence, whencompared to another composition of oligonucleotides having anothercomplementary common base sequence, provides higher cleavage rate of atranscript from the target nucleic acid sequence, and a cleavage patternwhich has only one major cleavage site, and the major cleavage site iswithin or close to a mutation or a SNP that defines the target sequencerelative to other similar sequences. In some embodiments, a mutation isa point mutation. In some embodiments, a major cleavage site is next toa mutation or a SNP that defines the target sequence relative to othersimilar sequences. In some embodiments, each common base sequence is100% complementary to the characteristic sequence element of the targetnucleic acid sequence. In some embodiments, a major cleavage site iswithin less than 5, 4, 3, or 1 internucleotidic linkage from a mutationor a SNP that defines the target sequence relative to other similarsequences. In some embodiments, a major cleavage site is within lessthan 5, 4, 3, or 1 internucleotidic linkage from a mutation or a SNPthat defines the target sequence relative to other similar sequences,and is within less than 5, 4, 3, or 1 internucleotidic linkage from acleavage site when a stereorandom composition of oligonucleotides havingthe same common sequence, and/or a composition of DNA oligonucleotideshaving the same common sequence, is used. In some embodiments, a majorcleavage site is a cleavage site when a stereorandom composition ofoligonucleotides having the same common sequence is used. In someembodiments, a major cleavage site is a major cleavage site when astereorandom composition of oligonucleotides having the same commonsequence is used. In some embodiments, a major cleavage site is acleavage site when a composition of DNA oligonucleotides having the samecommon sequence is used. In some embodiments, a major cleavage site is amajor cleavage site when a composition of DNA oligonucleotides havingthe same common sequence is used.

In some embodiments, when comparing effects of a first and a secondcommon base sequences, a stereorandom composition of oligonucleotideshaving a first common base sequence may be compared to a stereorandomcomposition of oligonucleotides having a second common base sequence. Insome embodiments, a stereorandom composition is a composition ofoligonucleotides having a common base sequence, a common pattern ofnucleoside modifications, and a common pattern of backbone linkages. Insome embodiments, a stereorandom composition is a composition ofoligonucleotides having a common base sequence, a common pattern ofnucleoside modifications, wherein each internucleotidic linkage isphosphorothioate. In some embodiments, when comparing effects of a firstand a second common base sequences, a chirally controlledoligonucleotide composition of oligonucleotides having a first commonbase sequence may be compared to a chirally controlled oligonucleotidecomposition of oligonucleotides having a second common base sequence. Insome embodiments, oligonucleotides in a chirally controlledoligonucleotide composition have a common base sequence, a commonpattern of nucleoside modifications, a common pattern of backbonelinkages, a common pattern of backbone chiral centers, and a commonpattern of backbone phosphorus modifications. In some embodiments, eachinternucleotidic linkage is phosphorothioate.

In some embodiments, oligonucleotide compositions and technologiesdescribed herein are particularly useful in the treatment ofHuntington's disease. For example, in some embodiments, the presentdisclosure defines stereochemically controlled oligonucleotidecompositions that direct cleavage (e.g., RNase H-mediated cleavage) ofnucleic acids associated with Huntington's disease. In some embodiments,such compositions direct preferential cleavage of a Huntington'sdisease-associated allele of a particular target sequence, relative toone or more (e.g., all non-Huntington's disease-associated) otheralleles of the sequence.

Huntington's disease is an inherited disease that can cause progressivedegeneration of nerve cells in the brain and affect a subject's motorand cognitive abilities. In some embodiments, Huntington's disease is anautosomal dominant disorder. In some embodiments, it is caused bymutations in the Huntingtin gene. Normal HTT gene contains 10 to 35 CAGtri-nucleotide repeats (SEQ ID NO: 1). People with 40 or more repeatsoften develop the disorder. In some embodiments, the expanded CAGsegment on the first exon of HTT gene leads to the production of anabnormally long version of the Huntingtin protein (expandedpolyglutamine tract) which is cut into smaller, toxic fragments thatbind together and accumulate in neurons, disrupting the normal functionsof these cells. Warby et al. (Am J Hum Genet. 2009, 84(3), 351-366)reported many SNPs that are associated with disease chromosomes and havestronger linkage associations with CAG expansion than those reportedbefore. Many SNPs highly associated with CAG expansion do not segregateindependently and are in Linkage Disequilibrium with each other. Amongother things, the present disclosure recognizes that strong associationbetween specific SNPs and CAG expanded chromosomes provides anattractive therapeutic opportunity for the treatment of HuntingtonDisease, e.g., through antisense therapy. Furthermore, the associationof specific SNPs combined with high rates of heterozygosity in HDpatients provides suitable targets for allele-specific knockdown of themutant gene product. For example references, see Liu et al. Journal ofHuntington's Disease 2, 2013, 491-500; Aronin, Neil and Pfister, EdithWO 2010/118263 A1; Pfister et al. Current Biology 2009, 19, 774-778.

In some embodiments, a targeted SNP of the present disclosure has highfrequency of heterozygosity in HD and has a particular variantassociated with the mutant HTT allele. In some embodiments, a SNP isrs362307. In some embodiments, a SNP is rs7685686. In some embodiments,a SNP may not be linked but may have a high heterozygous frequency. Insome embodiments, a SNP is rs362268 (3′-UTR region). In someembodiments, a SNP is rs362306 (3′-UTR region). In some embodiments, aSNP is rs2530595. In some embodiments, a SNP is rs362331.

In some embodiments, a provided method for treating or preventingHuntington's disease in a subject, comprising administering to thesubject a provided oligonucleotide compositions. In some embodiments, aprovided method for treating or preventing Huntington's disease in asubject, comprising administering to the subject a chirally controlledoligonucleotide composition comprising oligonucleotides of a particularoligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

In some embodiments, a provided method ameliorates a symptom ofHuntington's disease. In some embodiments, a provided method slows onsetof Huntington's disease. In some embodiments, a provided method slowsprogression of Huntington's disease.

In some embodiments, the present disclosure provides methods foridentifying patients for a given oligonucleotide composition. In someembodiments, the present disclosure provides methods for patientstratification. In some embodiments, a provided method comprisesidentifying a mutation and/or SNP associated with a disease-causingallele. For example, in some embodiments, a provided method comprisesidentifying in a subject a SNP associated with expanded CAG repeats thatare associated with or causing Huntington's disease.

In some embodiments, a subject has a SNP in the subject's Huntingtingene. In some embodiments, a subject has a SNP, wherein one allele ismutant Huntingtin associated with expanded CAG repeats. In someembodiments, a subject has a SNP selected from rs362307, rs7685686,rs362268, rs2530595, rs362331, or rs362306. In some embodiments,oligonucleotides of a provided composition have a sequence complementaryto a sequence comprising a SNP from the disease-causing allele (mutant),and the composition selectively suppresses expression from thediseasing-causing allele.

In some embodiments, the sequence of oligonucleotides in providedtechnologies (compounds, compositions, methods, etc.) comprises,consists of, or is the sequence of any oligonucleotide described herein.In some embodiments, a sequence is selected from Tables N1A, N2A, N3A,N4A or 8; or WV-1092, WVE120101, WV-2603 or WV-2595. In someembodiments, a sequence is selected from the sequence of WV-1092,WVE120101, WV-2603 or WV-2595. In some embodiments, providedoligonucleotides are of the type defined by WV-1092, WVE120101, WV-2603or WV-2595. In some embodiments, provided oligonucleotides are of thetype defined by WV-1092. In some embodiments, provided oligonucleotidesare of the type defined by WVE120101. In some embodiments, providedoligonucleotides are of the type defined by WV-2603. In someembodiments, provided oligonucleotides are of the type defined byWV-2595.

In some embodiments, provided oligonucleotide compositions comprises alipid and an oligonucleotide. In some embodiments, a lipid is conjugatedto an oligonucleotide.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from the list of: lauric acid, myristic acid, palmiticacid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid,gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid,arachidonic acid, and dilinoleyl. In some embodiments, a compositioncomprises an oligonucleotide and a lipid selected from the list of:lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid,linoleic acid, alpha-linolenic acid, gamma-linolenic acid,docosahexaenoic acid (cis-DHA), turbinaric acid, and dilinoleyl.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from:

In some embodiments, a composition comprises an oligonucleotide and alipid, wherein the lipid comprises a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group.

In some embodiments, an oligonucleotide composition comprises aplurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein one or more oligonucleotides of the plurality are individuallyconjugated to a lipid.

In some embodiments, a chirally controlled oligonucleotide compositioncomprises a plurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein:

the composition is chirally controlled in that the plurality ofoligonucleotides share the same stereochemistry at one or more chiralinternucleotidic linkages;

one or more oligonucleotides of the plurality are individuallyconjugated to a lipid; and one or more oligonucleotides of the pluralityare optionally and individually conjugated to a targeting compound ormoiety.

In some embodiments, a method of delivering an oligonucleotide to a cellor tissue in a human subject, comprises:

(a) Providing a composition of any one of the embodiments describedherein; and

(b) Administering the composition to the human subject such that theoligonucleotide is delivered to a cell or tissue in the subject.

In some embodiments, a method for delivering an oligonucleotide to acell or tissue comprises preparing a composition according to any one ofthe embodiments described herein and contacting the cell or tissue withthe composition.

In some embodiments, a method of modulating the level of a transcript orgene product of a gene in a cell, the method comprises the step ofcontacting the cell with a composition according to any one of theembodiments described herein, wherein the oligonucleotide is capable ofmodulating the level of the transcript or gene product.

In some embodiments, a method for inhibiting expression of a gene in acell or tissue comprises preparing a composition according to any one ofthe embodiments described herein and treating the cell or tissue withthe composition.

In some embodiments, a method for inhibiting expression of a gene in acell or tissue in a mammal comprises preparing a composition accordingto any one of the embodiments described herein and administering thecomposition to the mammal.

In some embodiments, a method of treating a disease that is caused bythe over-expression of one or several proteins in a cell or tissue in asubject, said method comprises the administration of a compositionaccording to any one of the embodiments described herein to the subject.

In some embodiments, a method of treating a disease that is caused by areduced, suppressed or missing expression of one or several proteins ina subject, said method comprises the administration of a compositionaccording to any one of the embodiments described herein to the subject.

In some embodiments, a method for generating an immune response in asubject, said method comprises the administration of a compositionaccording to any one of the embodiments described herein to the subject,wherein the biologically active compound is an immunomodulating nucleicacid.

In some embodiments, a method for treating a sign and/or symptom ofHuntington's Disease by providing a composition of any one of theembodiments described herein and administering the composition to thesubject.

In some embodiments, a method of modulating the amount ofRNaseH-mediated cleavage in a cell, the method comprises the step ofcontacting the cell with a composition according to any one of theembodiments described herein, wherein the oligonucleotide is capable ofmodulating the amount of RNaseH-mediated cleavage.

In some embodiments, a method of administering an oligonucleotide to asubject in need thereof, comprises steps of providing a compositioncomprises the agent a lipid, and administering the composition to thesubject, wherein the agent is any agent disclosed herein, and whereinthe lipid is any lipid disclosed herein.

In some embodiments, a method of treating a disease in a subject, themethod comprises steps of providing a composition comprises the agent alipid, and administering a therapeutically effective amount of thecomposition to the subject, wherein the agent is any agent disclosedherein, and wherein the lipid is any lipid disclosed herein, and whereinthe disease is any disease disclosed herein.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group.

In some embodiments, a lipid comprises an unsubstituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain.

In some embodiments, a lipid comprises two or more optionallysubstituted C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain.

In some embodiments, a lipid comprises no tricyclic or polycyclicmoiety.

In some embodiments, a lipid has the structure of R¹—COOH, wherein R¹ isan optionally substituted C₁₀-C₄₀ saturated or partially unsaturatedaliphatic chain.

The composition or method of any one of claim 16, wherein the lipid isconjugated through its carboxyl group.

The composition or method according to any one of the embodimentsdescribed herein, wherein the lipid is selected from:

In some embodiments, a lipid is conjugated to the oligonucleotide.

In some embodiments, a lipid is directly conjugated to theoligonucleotide.

In some embodiments, a lipid is conjugated to the oligonucleotide via alinker.

In some embodiments, a linker is selected from: an uncharged linker; acharged linker; a linker comprises an alkyl; a linker comprises aphosphate; a branched linker; an unbranched linker; a linker comprisesat least one cleavage group; a linker comprises at least one redoxcleavage group; a linker comprises at least one phosphate-based cleavagegroup; a linker comprises at least one acid-cleavage group; a linkercomprises at least one ester-based cleavage group; and a linkercomprises at least one peptide-based cleavage group.

In some embodiments, each oligonucleotide of the plurality isindividually conjugated to the same lipid at the same location.

In some embodiments, a lipid is conjugated to an oligonucleotide througha linker.

In some embodiments, one or more oligonucleotides of the plurality areindependently conjugated to a targeting compound or moiety.

In some embodiments, one or more oligonucleotides of the plurality areindependently conjugated to a lipid and a targeting compound or moiety.

In some embodiments, one or more oligonucleotides of the plurality areindependently conjugated to a lipid at one end and a targeting compoundor moiety at the other.

In some embodiments, oligonucleotides of the plurality share the samechemical modification patterns.

In some embodiments, oligonucleotides of the plurality share the samechemical modification patterns comprises one or more base modifications.

In some embodiments, oligonucleotides of the plurality share the samechemical modification patterns comprises one or more sugarmodifications.

In some embodiments, a common base sequence is capable of hybridizingwith a transcript in a cell, which transcript contains a mutation thatis linked to Huntington's Disease, or whose level, activity and/ordistribution is linked to Huntington's Disease.

In some embodiments, an oligonucleotide is a nucleic acid.

In some embodiments, an oligonucleotide is an oligonucleotide.

In some embodiments, an oligonucleotide is an oligonucleotide whichparticipates in RNaseH-mediated cleavage of a mutant Huntingtin genemRNA.

In some embodiments, a disease or disorder is Huntington's Disease.

In some embodiments, a lipid comprises an optionally substituted,C₁₀-C₈₀ saturated or partially unsaturated aliphatic group, wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—,—S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein each variable is independently as defined and describedherein.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted,C₁₀-C₆₀ saturated or partially unsaturated aliphatic group, wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—,—S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein each variable is independently as defined and describedherein.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted,C₁₀-C₄₀ saturated or partially unsaturated aliphatic group, wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—,—S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein each variable is independently as defined and describedherein.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain.

In some embodiments, a composition further comprises one or moreadditional components selected from: a polynucleotide, carbonicanhydrase inhibitor, a dye, an intercalating agent, an acridine, across-linker, psoralene, mitomycin C, a porphyrin, TPPC4, texaphyrin,Sapphyrin, a polycyclic aromatic hydrocarbon phenazine,dihydrophenazine, an artificial endonuclease, a chelating agent, EDTA,an alkylating agent, a phosphate, an amino, a mercapto, a PEG, PEG-40K,MPEG, [MPEG]₂, a polyamino, an alkyl, a substituted alkyl, aradiolabeled marker, an enzyme, a hapten biotin, a transport/absorptionfacilitator, aspirin, vitamin E, folic acid, a synthetic ribonuclease, aprotein, a glycoprotein, a peptide, a molecule having a specificaffinity for a co-ligand, an antibody, a hormone, a hormone receptor, anon-peptidic species, a lipid, a lectin, a carbohydrate, a vitamin, acofactor, selectivity agent, or a drug. In some embodiments, acomposition further comprises one or more additional components selectedfrom: a polynucleotide, carbonic anhydrase inhibitor, a dye, anintercalating agent, an acridine, a cross-linker, psoralene, mitomycinC, a porphyrin, TPPC4, texaphyrin, Sapphyrin, a polycyclic aromatichydrocarbon phenazine, dihydrophenazine, an artificial endonuclease, achelating agent, EDTA, an alkylating agent, a phosphate, an amino, amercapto, a PEG, PEG-40K, MPEG, [MPEG]₂, a polyamino, an alkyl, asubstituted alkyl, a radiolabeled marker, an enzyme, a hapten biotin, atransport/absorption facilitator, aspirin, vitamin E, folic acid, asynthetic ribonuclease, a protein, a glycoprotein, a peptide, a moleculehaving a specific affinity for a co-ligand, an antibody, a hormone, ahormone receptor, a non-peptidic species, a lipid, a lectin, acarbohydrate, a vitamin, a cofactor, or a drug.

In some embodiments, the present disclosure provides an oligonucleotideconjugated to a selectivity agent. In some embodiments, the presentdisclosure provides a composition comprising an oligonucleotide oroligonucleotide type comprising a selectivity agent. In someembodiments, a selectivity agent binds specifically to one or moreneurotransmitter transporters selected from the group consisting of adopamine transporter (DAT), a serotonin transporter (SERT), and anorepinephrine transporter (NET). In some embodiments, a selectivityagent is selected from the group consisting of a dopamine reuptakeinhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), anoradrenaline reuptake inhibitor (NRI), a norepinephrine-dopaminereuptake inhibitor (NDRI), and a serotonin-norepinephrine-dopaminereuptake inhibitor (SNDRI). In some embodiments, a selectivity agent isselected from the group consisting of a triple reuptake inhibitor, anoradrenaline dopamine double reuptake inhibitor, a serotonin singlereuptake inhibitor, a noradrenaline single reuptake inhibitor, and adopamine single reuptake inhibitor. In some embodiments, a selectivityagent is selected from the group consisting of a dopamine reuptakeinhibitor (DRI), a Norepinephrine-Dopamine Reuptake Inhibitor (NDRI) anda serotonin-Norepinephrine-Dopamine Reuptake Inhibitor (SNDRI). In someembodiments, a selectivity agent is selected from the selectivity agentswhich are described in U.S. Pat. Nos. 9,084,825; and 9,193,969; andWO2011131693, WO2014064258.

In some embodiments, a lipid comprises a C₁₀-C₈₀ linear, saturated orpartially unsaturated, aliphatic chain.

In some embodiments, a composition further comprises a linker linkingthe oligonucleotide and the lipid, wherein the linker is selected from:an uncharged linker; a charged linker; a linker comprises an alkyl; alinker comprises a phosphate; a branched linker; an unbranched linker; alinker comprises at least one cleavage group; a linker comprises atleast one redox cleavage group; a linker comprises at least onephosphate-based cleavage group; a linker comprises at least oneacid-cleavage group; a linker comprises at least one ester-basedcleavage group; a linker comprises at least one peptide-based cleavagegroup.

In some embodiments, an oligonucleotide comprises or consists of or isan oligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition.

In some embodiments, an oligonucleotide comprises or consists of or isan oligonucleotide composition or chirally controlled oligonucleotidecomposition, wherein the sequence of the oligonucleotide comprises orconsists of the sequence of any oligonucleotide described herein.

In some embodiments, an oligonucleotide comprises or consists of or isan oligonucleotide composition or chirally controlled oligonucleotidecomposition, wherein the sequence of the oligonucleotide comprises orconsists of the sequence of any oligonucleotide listed in Table 4.

In some embodiments, an oligonucleotide comprises or consists of or isan oligonucleotide composition or chirally controlled oligonucleotidecomposition, wherein the sequence of the oligonucleotide comprises orconsists of the sequence of a splice-switching oligonucleotide.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide is a chirally controlled oligonucleotidecomposition.

The composition or method of any of the embodiments described herein,wherein the disease or disorder is Huntington's Disease.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide comprises, consists of or is the sequence ofany oligonucleotide disclosed herein.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide is capable of differentiating between awild-type and a mutant Huntingtin allele.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.

The composition or method of any of the embodiments described herein,wherein the oligonucleotide comprises, consists of or is the sequence ofany oligonucleotide disclosed in Table 4.

In some embodiments, an oligonucleotide comprises or consists of or isan oligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of any of: WV-1092, WV-2595, orWV-2603.

In some embodiments, a sequence of an oligonucleotide includes any oneor more of: base sequence (including length); pattern of chemicalmodifications to sugar and base moieties; pattern of backbone linkages;pattern of natural phosphate linkages, phosphorothioate linkages,phosphorothioate triester linkages, and combinations thereof; pattern ofbackbone chiral centers; pattern of stereochemistry (Rp/Sp) of chiralinternucleotidic linkages; pattern of backbone phosphorus modifications;pattern of modifications on the internucleotidic phosphorus atom, suchas —S⁻, and -L-R¹ of formula I.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type, wherein the oligonucleotides target amutant Huntingtin gene, and the length is from about 10 to about 50nucleotides, wherein the backbone linkages comprise at least onephosphorothioate, and wherein the pattern of backbone chiral centerscomprises at least one chiral center in a Rp conformation and at leastone chiral center in a Sp conformation.

In some embodiments, the present disclosure provides a method forcleavage of a nucleic acid having a base sequence comprising a targetsequence, the method comprising steps of:

(a) contacting a nucleic acid having a base sequence comprising a targetsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to the        target sequence in the nucleic acid;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type,        wherein the oligonucleotide targets a mutant Huntingtin gene,        and the length is from about 10 to about 50 nucleotides, wherein        the backbone linkages comprise at least one phosphorothioate,        and wherein the pattern of backbone chiral centers comprises at        least one chiral center in a Rp conformation and at least one        chiral center in a Sp conformation.

In some embodiments, the present disclosure provides a method forcleavage of a nucleic acid having a base sequence comprising a targetsequence, the method comprising steps of:

(a) contacting a nucleic acid having a base sequence comprising a targetsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to the        target sequence in the nucleic acid;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type,        wherein the oligonucleotide targets a mutant Huntingtin gene,        and the length is from about 10 to about 50 nucleotides, wherein        the backbone linkages comprise at least one phosphorothioate,        and wherein the pattern of backbone chiral centers comprises at        least one chiral center in a Rp conformation and at least one        chiral center in a Sp conformation; and

(b) cleavage of the nucleic acid mediated by a RNAseH or RNAinterference mechanism.

In some embodiments, a provided composition further comprises aselectivity agent selected from: the group of compounds which bindsspecifically to one or more neurotransmitter transporters selected fromthe group consisting of a dopamine transporter (DAT), a serotonintransporter (SERT), and a norepinephrine transporter (NET); the groupconsisting of a dopamine reuptake inhibitor (DRI), a selective serotoninreuptake inhibitor (SSRI), a noradrenaline reuptake inhibitor (NRI), anorepinephrine-dopamine reuptake inhibitor (NDRI), and aserotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI); the groupconsisting of a triple reuptake inhibitor, a noradrenaline dopaminedouble reuptake inhibitor, a serotonin single reuptake inhibitor, anoradrenaline single reuptake inhibitor, and a dopamine single reuptakeinhibitor; and the group consisting of a dopamine reuptake inhibitor(DRI), a Norepinephrine-Dopamine Reuptake Inhibitor (NDRI) and aserotonin-Norepinephrine-Dopamine Reuptake Inhibitor (SNDRI).

In some embodiments, a provided composition comprises oligonucleotideswherein the base sequence, pattern of backbone linkages and/or patternof backbone chiral centers of the oligonucleotides comprises or consistsof the base sequence, pattern of backbone linkages and/or pattern ofbackbone chiral centers of any of any oligonucleotide selected fromTables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

In some embodiments, a provided composition comprises oligonucleotideswherein the base sequence, pattern of backbone linkages and/or patternof backbone chiral centers of the oligonucleotides comprises or consistsof the base sequence, and pattern of backbone linkages, and/or patternof backbone chiral centers of any of any oligonucleotide selected fromTables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

In some embodiments, a provided composition comprises oligonucleotideswherein the base sequence, pattern of backbone linkages and/or patternof backbone chiral centers of the oligonucleotides comprises or consistsof the base sequence, and pattern of backbone linkages, and pattern ofbackbone chiral centers of any of any oligonucleotide selected fromTables N1A, N2A, N3A, N4A and 8; and WV-1092, WV-2595, and WV-2603.

Definitions

Aliphatic: The term “aliphatic” or “aliphatic group”, as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic or polycyclic hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic (also referred to herein as “carbocycle” “cycloaliphatic” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule. In some embodiments, aliphatic groups contain 1-50 aliphaticcarbon atoms. Unless otherwise specified, aliphatic groups contain 1-10aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-6 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic or bicyclic C₃-C₁₀ hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic, that has a single point of attachment to the rest ofthe molecule. In some embodiments, “cycloaliphatic” (or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkylene: The term “alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein nis a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3,from 1 to 2, or from 2 to 3. A substituted alkylene chain is apolymethylene group in which one or more methylene hydrogen atoms arereplaced with a substituent. Suitable substituents include thosedescribed below for a substituted aliphatic group.

Alkenylene: The term “alkenylene” refers to a bivalent alkenyl group. Asubstituted alkenylene chain is a polymethylene group containing atleast one double bond in which one or more hydrogen atoms are replacedwith a substituent. Suitable substituents include those described belowfor a substituted aliphatic group.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, a genetically-engineered animal,and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means±5 mg/kg/day.

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring.” In certainembodiments of the present disclosure, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, 20 or more amino acids are requiredto be characteristic of a protein. In general, a characteristic portionis one that, in addition to the sequence identity specified above,shares at least one functional characteristic with the relevant intactprotein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Characteristic structural element: The term “characteristic structuralelement” refers to a distinctive structural element (e.g., corestructure, collection of pendant moieties, sequence element, etc) thatis found in all members of a family of polypeptides, small molecules, ornucleic acids, and therefore can be used by those of ordinary skill inthe art to define members of the family.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions or circumstances that are sufficiently similarto one another to permit comparison of results obtained or phenomenaobserved. In some embodiments, comparable sets of conditions orcircumstances are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Thoseof ordinary skill in the art will appreciate that sets of conditions arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder the different sets of conditions or circumstances are caused by orindicative of the variation in those features that are varied.

Dosing regimen: As used herein, a “dosing regimen” or “therapeuticregimen” refers to a set of unit doses (typically more than one) thatare administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, all doses within a dosing regimen are of thesame unit dose amount. In some embodiments, different doses within adosing regimen are of different amounts. In some embodiments, a dosingregimen comprises a first dose in a first dose amount, followed by oneor more additional doses in a second dose amount different from thefirst dose amount. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount same as the first dose amount.

Equivalent agents: Those of ordinary skill in the art, reading thepresent disclosure, will appreciate that the scope of useful agents inthe context of the present disclosure is not limited to thosespecifically mentioned or exemplified herein. In particular, thoseskilled in the art will recognize that active agents typically have astructure that consists of a core and attached pendant moieties, andfurthermore will appreciate that simple variations of such core and/orpendant moieties may not significantly alter activity of the agent. Forexample, in some embodiments, substitution of one or more pendantmoieties with groups of comparable three-dimensional structure and/orchemical reactivity characteristics may generate a substituted compoundor portion equivalent to a parent reference compound or portion. In someembodiments, addition or removal of one or more pendant moieties maygenerate a substituted compound equivalent to a parent referencecompound. In some embodiments, alteration of core structure, for exampleby addition or removal of a small number of bonds (typically not morethan 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generatea substituted compound equivalent to a parent reference compound. Inmany embodiments, equivalent compounds may be prepared by methodsillustrated in general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional or provided synthesis procedures.In these reactions, it is also possible to make use of variants, whichare in themselves known, but are not mentioned here.

Equivalent Dosage: The term “equivalent dosage” is used herein tocompare dosages of different pharmaceutically active agents that effectthe same biological result. Dosages of two different agents areconsidered to be “equivalent” to one another in accordance with thepresent disclosure if they achieve a comparable level or extent of thebiological result. In some embodiments, equivalent dosages of differentpharmaceutical agents for use in accordance with the present disclosureare determined using in vitro and/or in vivo assays as described herein.In some embodiments, one or more lysosomal activating agents for use inaccordance with the present disclosure is utilized at a dose equivalentto a dose of a reference lysosomal activating agent; in some suchembodiments, the reference lysosomal activating agent for such purposeis selected from the group consisting of small molecule allostericactivators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine),antioxidants (e.g., n-acetyl-cysteine), and regulators of cellulartrafficking (e.g., Rab1a polypeptide).

Heteroaliphatic: The term “heteroaliphatic” refers to an aliphatic groupwherein one or more units selected from C, CH, CH₂, or CH₃ areindependently replaced by a heteroatom. In some embodiments, aheteroaliphatic group is heteroalkyl. In some embodiments, aheteroaliphatic group is heteroalkenyl.

Heteroaryl: The terms “heteroaryl” and “heteroar-,” used alone or aspart of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,”refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ringatoms; having 6, 10, or 14π electrons shared in a cyclic array; andhaving, in addition to carbon atoms, from one to five heteroatoms. Theterm “heteroatom” refers to nitrogen, oxygen, or sulfur, and includesany oxidized form of nitrogen or sulfur, and any quaternized form of abasic nitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-,” as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur,nitrogen, phosphorus, boron, selenium, or silicon (including, anyoxidized form of nitrogen, boron, selenium, sulfur, phosphorus, orsilicon; the quaternized form of any basic nitrogen or; a substitutablenitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl)).

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a stable 3- to 7-membered monocyclic or 7-10-memberedbicyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understoodmeaning referring to administration of a compound or composition intothe peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C₁₋₄ straight orbranched alkyl group. Example lower alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C₁₋₄ straight orbranched alkyl group that is substituted with one or more halogen atoms.

Optionally substituted: As described herein, compounds of the disclosuremay contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis disclosure are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(◯); —(CH₂)₀₋₄OR^(◯); —O(CH₂)₀₋₄R^(◯), —O—(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄CH(OR^(◯))₂; —(CH₂)₀₋₄SR^(◯); —(CH₂)₀₋₄Ph, which may besubstituted with R^(◯); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(◯); —CH═CHPh, which may be substituted with R^(◯);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(◯); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(◯))₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯);—N(R^(◯))C(S)R^(◯); —(CH₂)₀₋₄N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))C(S)NR^(◯)₂; —(CH₂)₀₋₄N(R^(◯))C(O)OR^(◯); —N(R^(◯))N(R^(◯))C(O)R^(◯);—N(R^(◯))N(R^(◯))C(O)NR^(◯) ₂; —N(R^(◯))N(R^(◯))C(O)OR^(◯);—(CH₂)₀₋₄C(O)R^(◯); —C(S)R^(◯); —(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄C(O)SR^(◯); —(CH₂)₀₋₄C(O)OSiR^(◯) ₃; —(CH₂)₀₋₄OC(O)R^(◯);—OC(O)(CH₂)₀₋₄SR, —SC(S)SR^(◯); —(CH₂)₀₋₄SC(O)R^(◯); —(CH₂)₀₋₄C(O)NR^(◯)₂; —C(S)NR^(◯) ₂; —C(S)SR^(◯); —SC(S)SR^(◯), —(CH₂)₀₋₄OC(O)NR^(◯) ₂;—C(O)N(OR^(◯))R^(◯); —C(O)C(O)R^(◯); —C(O)CH₂C(O)R^(◯);—C(NOR^(◯))R^(◯); —(CH₂)₀₋₄SSR^(◯); —(CH₂)₀₋₄S(O)₂R^(◯);—(CH₂)₀₋₄S(O)₂OR^(◯); —(CH₂)₀₋₄OS(O)₂R^(◯); —S(O)₂NR^(◯) ₂; —(CH₂)₀₋₄S(O)R^(◯); —N(R^(◯))S(O)₂NR^(◯) ₂; —N(R^(◯))S(O)₂R^(◯); —N(OR^(◯))R^(◯);—C(NH)NR^(◯) ₂; —P(O)₂R^(◯); —P(O)R^(◯) ₂; —OP(O)R^(◯) ₂;—OP(O)(OR^(◯))₂; —SiR^(◯) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(◯))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(◯))₂, wherein each R^(◯) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(◯), taken together with their intervening atom(s), form a 3-12membered saturated, partially unsaturated, or aryl mono- or bicyclicring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(◯) (or the ring formed by takingtwo independent occurrences of R^(◯) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(°) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R)S(O)₂R^(†); wherein each R^(†) isindependently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12 membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal, and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). In some embodiments, pharmaceuticallyacceptable salt include, but are not limited to, nontoxic acid additionsalts, which are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. In someembodiments, pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. In someembodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

Prodrug: A general, a “prodrug,” as that term is used herein and as isunderstood in the art, is an entity that, when administered to anorganism, is metabolized in the body to deliver an active (e.g.,therapeutic or diagnostic) agent of interest. Typically, such metabolisminvolves removal of at least one “prodrug moiety” so that the activeagent is formed. Various forms of “prodrugs” are known in the art. Forexamples of such prodrug moieties, see:

-   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and    Methods in Enzymology, 42:309-396, edited by K. Widder, et al.    (Academic Press, 1985);-   b) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,    2011);-   c) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,    2011);-   d) A Textbook of Drug Design and Development, edited by    Krogsgaard-Larsen;-   e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H.    Bundgaard, p. 113-191 (1991);-   f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);-   g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285    (1988); and-   h) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

As with other compounds described herein, prodrugs may be provided inany of a variety of forms, e.g., crystal forms, salt forms etc. In someembodiments, prodrugs are provided as pharmaceutically acceptable saltsthereof.

Protecting group: The term “protecting group,” as used herein, is wellknown in the art and includes those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference. Also included are those protecting groups speciallyadapted for nucleoside and nucleotide chemistry described in CurrentProtocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al.06/2012, the entirety of Chapter 2 is incorporated herein by reference.Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methyl sulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limitedto, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylicacids. Examples of suitable silyl groups include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triisopropylsilyl, and the like. Examples of suitable alkyl groupsinclude methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl,t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groupsinclude allyl. Examples of suitable aryl groups include optionallysubstituted phenyl, biphenyl, or naphthyl. Examples of suitablearylalkyl groups include optionally substituted benzyl (e.g.,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In some embodiments, a hydroxyl protecting group is acetyl, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethyl silylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl,trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate,tosylate, triflate, trityl, monomethoxytrityl (MMTr),4,4′-dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr),2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl(NPE), 2-(4-nitrophenyl sulfonyl)ethyl, 3,5-dichlorophenyl,2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl,2-(2-nitrophenyl)ethyl, butylthiocarbonyl,4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl,2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl(Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl(MOX). In some embodiments, each of the hydroxyl protecting groups is,independently selected from acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, thehydroxyl protecting group is selected from the group consisting oftrityl, monomethoxytrityl and 4,4′-dimethoxytrityl group.

In some embodiments, a phosphorus protecting group is a group attachedto the internucleotide phosphorus linkage throughout oligonucleotidesynthesis. In some embodiments, the phosphorus protecting group isattached to the sulfur atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorus protecting group isattached to the oxygen atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorus protecting group isattached to the oxygen atom of the internucleotide phosphate linkage. Insome embodiments the phosphorus protecting group is 2-cyanoethyl (CE orCne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl,benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe),2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl,4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl,3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl,2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). In some embodiments, proteins include onlynaturally-occurring amino acids. In some embodiments, proteins includeone or more non-naturally-occurring amino acids (e.g., moieties thatform one or more peptide bonds with adjacent amino acids). In someembodiments, one or more residues in a protein chain contain anon-amino-acid moiety (e.g., a glycan, etc). In some embodiments, aprotein includes more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. In someembodiments, proteins contain L-amino acids, D-amino acids, or both; insome embodiments, proteins contain one or more amino acid modificationsor analogs known in the art. Useful modifications include, e.g.,terminal acetylation, amidation, methylation, etc. The term “peptide” isgenerally used to refer to a polypeptide having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids. In some embodiments, proteinsare antibodies, antibody fragments, biologically active portionsthereof, and/or characteristic portions thereof.

Sample: A “sample” as used herein is a specific organism or materialobtained therefrom. In some embodiments, a sample is a biological sampleobtained or derived from a source of interest, as described herein. Insome embodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample isor comprises any one or more of: bone marrow; blood; blood cells;ascites; tissue or fine needle biopsy samples; cell-containing bodyfluids; free floating nucleic acids; sputum; saliva; urine;cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph;gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasalswabs; washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; feces, other body fluids, secretions,and/or excretions; and/or cells therefrom, etc. In some embodiments, abiological sample is or comprises cells obtained from an individual. Insome embodiments, a sample is a “primary sample” obtained directly froma source of interest by any appropriate means. For example, in someembodiments, a primary biological sample is obtained by methods selectedfrom the group consisting of biopsy (e.g., fine needle aspiration ortissue biopsy), surgery, collection of body fluid (e.g., blood, lymph,feces etc.), etc. In some embodiments, as will be clear from context,the term “sample” refers to a preparation that is obtained by processing(e.g., by removing one or more components of and/or by adding one ormore agents to) a primary sample. For example, filtering using asemi-permeable membrane. Such a “processed sample” may comprise, forexample nucleic acids or proteins extracted from a sample or obtained bysubjecting a primary sample to techniques such as amplification orreverse transcription of mRNA, isolation and/or purification of certaincomponents, etc. In some embodiments, a sample is an organism. In someembodiments, a sample is a plant. In some embodiments, a sample is ananimal. In some embodiments, a sample is a human. In some embodiments, asample is an organism other than a human.

Stereochemically isomeric forms: The phrase “stereochemically isomericforms,” as used herein, refers to different compounds made up of thesame atoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable. In someembodiments of the disclosure, provided chemical compositions may be orinclude pure preparations of individual stereochemically isomeric formsof a compound; in some embodiments, provided chemical compositions maybe or include mixtures of two or more stereochemically isomeric forms ofthe compound. In certain embodiments, such mixtures contain equalamounts of different stereochemically isomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent stereochemically isomeric forms. In some embodiments, achemical composition may contain all diastereomers and/or enantiomers ofthe compound. In some embodiments, a chemical composition may containless than all diastereomers and/or enantiomers of a compound. In someembodiments, if a particular enantiomer of a compound of the presentdisclosure is desired, it may be prepared, for example, by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, diastereomeric saltsare formed with an appropriate optically-active acid, and resolved, forexample, by fractional crystallization.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present disclosure e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present disclosure. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of thedisclosure, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments of the disclosure, chemical compositions may be provided asmixtures of two or more tautomeric forms of a compound. In certainembodiments, such mixtures contain equal amounts of different tautomericforms; in certain embodiments, such mixtures contain different amountsof at least two different tautomeric forms of a compound. In someembodiments of the disclosure, chemical compositions may contain alltautomeric forms of a compound. In some embodiments of the disclosure,chemical compositions may contain less than all tautomeric forms of acompound. In some embodiments of the disclosure, chemical compositionsmay contain one or more tautomeric forms of a compound in amounts thatvary over time as a result of interconversion. In some embodiments ofthe disclosure, the tautomerism is keto-enol tautomerism. One of skillin the chemical arts would recognize that a keto-enol tautomer can be“trapped” (i.e., chemically modified such that it remains in the “enol”form) using any suitable reagent known in the chemical arts in toprovide an enol derivative that may subsequently be isolated using oneor more suitable techniques known in the art. Unless otherwiseindicated, the present disclosure encompasses all tautomeric forms ofrelevant compounds, whether in pure form or in admixture with oneanother.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unsaturated: The term “unsaturated,” as used herein, means that a moietyhas one or more units of unsaturation.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Wild-type: As used herein, the term “wild-type” has its art-understoodmeaning that refers to an entity having a structure and/or activity asfound in nature in a “normal” (as contrasted with mutant, diseased,altered, etc) state or context. Those of ordinary skill in the art willappreciate that wild type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Nucleic acid: The term “nucleic acid” includes any nucleotides, modifiedvariants thereof, analogs thereof, and polymers thereof. The term“polynucleotide” as used herein refer to a polymeric form of nucleotidesof any length, either ribonucleotides (RNA) or deoxyribonucleotides(DNA) or modified variants or analogs thereof. These terms refer to theprimary structure of the molecules and, thus, include double- andsingle-stranded DNA, and double- and single-stranded RNA. These termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated, protected and/or capped nucleotides orpolynucleotides. The terms encompass poly- or oligo-ribonucleotides(RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derivedfrom N-glycosides or C-glycosides of nucleobases and/or modifiednucleobases; nucleic acids derived from sugars and/or modified sugars;and nucleic acids derived from phosphate bridges and/or modifiedphosphorus-atom bridges (also referred to herein as “internucleotidelinkages”). The term encompasses nucleic acids containing anycombinations of nucleobases, modified nucleobases, sugars, modifiedsugars, phosphate bridges or modified phosphorus atom bridges. Examplesinclude, and are not limited to, nucleic acids containing ribosemoieties, the nucleic acids containing deoxy-ribose moieties, nucleicacids containing both ribose and deoxyribose moieties, nucleic acidscontaining ribose and modified ribose moieties. The prefix poly-refersto a nucleic acid containing 2 to about 10,000 nucleotide monomer unitsand wherein the prefix oligo-refers to a nucleic acid containing 2 toabout 200 nucleotide monomer units.

Nucleotide: The term “nucleotide” as used herein refers to a monomericunit of a polynucleotide that consists of a heterocyclic base, a sugar,and one or more phosphate groups or phosphorus-containinginternucleotidic linkages. The naturally occurring bases, (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) arederivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleotides are linked via internucleotidiclinkages to form nucleic acids, or polynucleotides. Manyinternucleotidic linkages are known in the art (such as, though notlimited to, phosphate, phosphorothioates, boranophosphates and thelike). Artificial nucleic acids include PNAs (peptide nucleic acids),phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates,boranophosphates, methylphosphonates, phosphonoacetates,thiophosphonoacetates and other variants of the phosphate backbone ofnative nucleic acids, such as those described herein. Other analogs(e.g., artificial nucleic acids or components which can be incorporatedinto a nucleic acid or artificial nucleic acid) include: boranophosphateRNA, FANA, locked nucleic acids (LNA), Morpholinos, peptidic nucleicacids (PNA), threose nucleic acid (TNA), and glycol nucleic acid (GNA).These skilled in the art are aware of a variety of modified nucleotidesor nucleotide analogs, including, for example, those described in anyof: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrixet al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem.4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem.1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273;Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo.Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5:530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256;Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Morita etal. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo.Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett.2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett.39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen etal. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun.1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al.2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53:8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al.2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc.Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R;Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth;Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From NucleicAcids Symposium Series (2008), 52(1), 553-554; Singh et al. 1998 Chem.Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh etal. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm.2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; VanAerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur etal. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071;or WO 2016/079181.

Nucleoside: The term “nucleoside” refers to a moiety wherein anucleobase or a modified nucleobase is covalently bound to a sugar ormodified sugar.

Sugar: The term “sugar” refers to a monosaccharide in closed and/or openform. Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties. As used herein,the term also encompasses structural analogs used in lieu ofconventional sugar molecules, such as glycol, polymer of which forms thebackbone of the nucleic acid analog, glycol nucleic acid (“GNA”).

Modified sugar: The term “modified sugar” refers to a moiety that canreplace a sugar. The modified sugar mimics the spatial arrangement,electronic properties, or some other physicochemical property of asugar.

Nucleobase: The term “nucleobase” refers to the parts of nucleic acidsthat are involved in the hydrogen-bonding that binds one nucleic acidstrand to another complementary strand in a sequence specific manner.The most common naturally-occurring nucleobases are adenine (A), guanine(G), uracil (U), cytosine (C), and thymine (T). In some embodiments, thenaturally-occurring nucleobases are modified adenine, guanine, uracil,cytosine, or thymine. In some embodiments, the naturally-occurringnucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, a nucleobase is a “modified nucleobase,”e.g., a nucleobase other than adenine (A), guanine (G), uracil (U),cytosine (C), and thymine (T). In some embodiments, the modifiednucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, the modified nucleobase mimics the spatialarrangement, electronic properties, or some other physicochemicalproperty of the nucleobase and retains the property of hydrogen-bondingthat binds one nucleic acid strand to another in a sequence specificmanner. In some embodiments, a modified nucleobase can pair with all ofthe five naturally occurring bases (uracil, thymine, adenine, cytosine,or guanine) without substantially affecting the melting behavior,recognition by intracellular enzymes or activity of the oligonucleotideduplex.

Chiral ligand: The term “chiral ligand” or “chiral auxiliary” refers toa moiety that is chiral and can be incorporated into a reaction so thatthe reaction can be carried out with certain stereoselectivity.

Condensing reagent: In a condensation reaction, the term “condensingreagent” refers to a reagent that activates a less reactive site andrenders it more susceptible to attack by another reagent. In someembodiments, such another reagent is a nucleophile.

Blocking group: The term “blocking group” refers to a group that masksthe reactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking group. In someembodiments, a blocking group is a protecting group.

Moiety: The term “moiety” refers to a specific segment or functionalgroup of a molecule. Chemical moieties are often recognized chemicalentities embedded in or appended to a molecule.

Solid support: The term “solid support” refers to any support whichenables synthesis of nucleic acids. In some embodiments, the term refersto a glass or a polymer, that is insoluble in the media employed in thereaction steps performed to synthesize nucleic acids, and is derivatizedto comprise reactive groups. In some embodiments, the solid support isHighly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). Insome embodiments, the solid support is Controlled Pore Glass (CPG). Insome embodiments, the solid support is hybrid support of Controlled PoreGlass (CPG) and Highly Cross-linked Polystyrene (HCP).

Linking moiety: The term “linking moiety” refers to any moietyoptionally positioned between the terminal nucleoside and the solidsupport or between the terminal nucleoside and another nucleoside,nucleotide, or nucleic acid.

DNA molecule: A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form or a double-stranded helix. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences can be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

Coding sequence: A DNA “coding sequence” or “coding region” is adouble-stranded DNA sequence which is transcribed and translated into apolypeptide in vivo when placed under the control of appropriateexpression control sequences. The boundaries of the coding sequence (the“open reading frame” or “ORF”) are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequencesfrom eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence is,usually, be located 3′ to the coding sequence. The term “non-codingsequence” or “non-coding region” refers to regions of a polynucleotidesequence that are not translated into amino acids (e.g. 5′ and 3′un-translated regions).

Reading frame: The term “reading frame” refers to one of the sixpossible reading frames, three in each direction, of the double strandedDNA molecule. The reading frame that is used determines which codons areused to encode amino acids within the coding sequence of a DNA molecule.

Antisense: As used herein, an “antisense” nucleic acid moleculecomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule, complementary to an mRNAsequence or complementary to the coding strand of a gene. Accordingly,an antisense nucleic acid molecule can associate via hydrogen bonds to asense nucleic acid molecule. In some embodiments, an antisenseoligonucleotide is an oligonucleotide which participates inRNaseH-mediated cleavage; for example, an antisense oligonucleotidehybridizes in a sequence-specific manner to a portion of a target mRNA,thus targeting the mRNA for cleavage by RNaseH. In some embodiments, anantisense oligonucleotide is able to differentiate between a wild-typeand a mutant allele of a target. In some embodiments, an antisenseoligonucleotide significantly participates in RNaseH-mediated cleavageof a mutant allele but participates in RNaseH-mediated cleavage of awild-type allele to a much less degree (e.g., does not significantlyparticipate in RNaseH-mediated cleavage of the wild-type allele of thetarget).

Wobble position: As used herein, a “wobble position” refers to the thirdposition of a codon. Mutations in a DNA molecule within the wobbleposition of a codon, in some embodiments, result in silent orconservative mutations at the amino acid level. For example, there arefour codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thusmutation of any wobble position nucleotide, to any other nucleotideselected from A, U, C and G, does not result in a change at the aminoacid level of the encoded protein and, therefore, is a silentsubstitution.

Silent substitution: a “silent substitution” or “silent mutation” is onein which a nucleotide within a codon is modified, but does not result ina change in the amino acid residue encoded by the codon. Examplesinclude mutations in the third position of a codon, as well in the firstposition of certain codons such as in the codon “CGG” which, whenmutated to AGG, still encodes Arg.

Gene: The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein or a portion thereof. The DNA molecule can contain anopen reading frame encoding the protein (as exon sequences) and canfurther include intron sequences. The term “intron” as used herein,refers to a DNA sequence present in a given gene which is not translatedinto protein and is found in some, but not all cases, between exons. Itcan be desirable for the gene to be operably linked to, (or it cancomprise), one or more promoters, enhancers, repressors and/or otherregulatory sequences to modulate the activity or expression of the gene,as is well known in the art.

Complementary DNA: As used herein, a “complementary DNA” or “cDNA”includes recombinant polynucleotides synthesized by reversetranscription of mRNA and from which intervening sequences (introns)have been removed.

Homology: “Homology” or “identity” or “similarity” refers to sequencesimilarity between two nucleic acid molecules. Homology and identity caneach be determined by comparing a position in each sequence which can bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar nucleic acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar nucleic acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, less than 35% identity, less than 30% identity,or less than 25% identity with a sequence described herein. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

In some embodiments, the term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes with similar functions or motifs. The nucleic acid sequencesdescribed herein can be used as a “query sequence” to perform a searchagainst public databases, for example, to identify other family members,related sequences or homologs. In some embodiments, such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments,BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous tonucleic acid molecules of the disclosure. In some embodiments, to obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

Identity: As used herein, “identity” means the percentage of identicalnucleotide residues at corresponding positions in two or more sequenceswhen the sequences are aligned to maximize sequence matching, i.e.,taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well-known Smith Watermanalgorithm can also be used to determine identity.

Heterologous: A “heterologous” region of a DNA sequence is anidentifiable segment of DNA within a larger DNA sequence that is notfound in association with the larger sequence in nature. Thus, when theheterologous region encodes a mammalian gene, the gene can usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous codingsequence is a sequence where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains intronsor synthetic sequences having codons or motifs different than theunmodified gene). Allelic variations or naturally-occurring mutationalevents do not give rise to a heterologous region of DNA as definedherein.

Transition mutation: The term “transition mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by another pyrimidine, or a purine (adenosine(A) or guanosine (G) is replaced by another purine.

Transversion mutation: The term “transversion mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by a purine (adenosine (A) or guanosine (G),or a purine is replaced by a pyrimidine.

Oligonucleotide: the term “oligonucleotide” refers to a polymer oroligomer of nucleotide monomers, containing any combination ofnucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges, or modified phosphorus atom bridges (also referred to herein as“internucleotidic linkage”, defined further herein).

Oligonucleotides can be single-stranded or double-stranded. As usedherein, the term “oligonucleotide strand” encompasses a single-strandedoligonucleotide. A single-stranded oligonucleotide can havedouble-stranded regions and a double-stranded oligonucleotide can havesingle-stranded regions. Example oligonucleotides include, but are notlimited to structural genes, genes including control and terminationregions, self-replicating systems such as viral or plasmid DNA,single-stranded and double-stranded siRNAs and other RNA interferencereagents (RNAi agents or iRNA agents), shRNA, antisenseoligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, Ul adaptors, triplex-formingoligonucleotides, G-quadruplex oligonucleotides, RNA activators,immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Double-stranded and single-stranded oligonucleotides that are effectivein inducing RNA interference are also referred to as siRNA, RNAi agent,or iRNA agent, herein. In some embodiments, these RNA interferenceinducing oligonucleotides associate with a cytoplasmic multi-proteincomplex known as RNAi-induced silencing complex (RISC). In manyembodiments, single-stranded and double-stranded RNAi agents aresufficiently long that they can be cleaved by an endogenous molecule,e.g., by Dicer, to produce smaller oligonucleotides that can enter theRISC machinery and participate in RISC mediated cleavage of a targetsequence, e.g. a target mRNA.

Oligonucleotides of the present disclosure can be of various lengths. Inparticular embodiments, oligonucleotides can range from about 2 to about200 nucleotides in length. In various related embodiments,oligonucleotides, single-stranded, double-stranded, and triple-stranded,can range in length from about 4 to about 10 nucleotides, from about 10to about 50 nucleotides, from about 20 to about 50 nucleotides, fromabout 15 to about 30 nucleotides, from about 20 to about 30 nucleotidesin length. In some embodiments, the oligonucleotide is from about 9 toabout 39 nucleotides in length. In some embodiments, the oligonucleotideis at least 4 nucleotides in length. In some embodiments, theoligonucleotide is at least 5 nucleotides in length. In someembodiments, the oligonucleotide is at least 6 nucleotides in length. Insome embodiments, the oligonucleotide is at least 7 nucleotides inlength. In some embodiments, the oligonucleotide is at least 8nucleotides in length. In some embodiments, the oligonucleotide is atleast 9 nucleotides in length. In some embodiments, the oligonucleotideis at least 10 nucleotides in length. In some embodiments, theoligonucleotide is at least 11 nucleotides in length. In someembodiments, the oligonucleotide is at least 12 nucleotides in length.In some embodiments, the oligonucleotide is at least 15 nucleotides inlength. In some embodiments, the oligonucleotide is at least 20nucleotides in length. In some embodiments, the oligonucleotide is atleast 25 nucleotides in length. In some embodiments, the oligonucleotideis at least 30 nucleotides in length. In some embodiments, theoligonucleotide is a duplex of complementary strands of at least 18nucleotides in length. In some embodiments, the oligonucleotide is aduplex of complementary strands of at least 21 nucleotides in length.

Internucleotidic linkage. As used herein, the phrase “internucleotidiclinkage” refers generally to the phosphorus-containing linkage betweennucleotide units of an oligonucleotide, and is interchangeable with“inter-sugar linkage” and “phosphorus atom bridge,” as used above andherein. In some embodiments, an internucleotidic linkage is aphosphodiester linkage, as found in naturally occurring DNA and RNAmolecules. In some embodiments, an internucleotidic linkage is a“modified internucleotidic linkage” wherein each oxygen atom of thephosphodiester linkage is optionally and independently replaced by anorganic or inorganic moiety. In some embodiments, such an organic orinorganic moiety is selected from but not limited to ═S, ═Se, ═NR′,—SR′, —SeR′, —N(R′)₂, B(R′)₃, —S—, —Se—, and —N(R′)—, wherein each R′ isindependently as defined and described below. In some embodiments, aninternucleotidic linkage is a phosphotriester linkage, phosphorothioatediester linkage

or modified phosphorothioate triester linkage. It is understood by aperson of ordinary skill in the art that the internucleotidic linkagemay exist as an anion or cation at a given pH due to the existence ofacid or base moieties in the linkage.

Unless otherwise specified, when used with an oligonucleotide sequence,each of s, s1, s2, s3, s4, s5, s6 and s7 independently represents thefollowing modified internucleotidic linkage as illustrated in Table 1,below.

TABLE 1 Example Modified Internucleotidic Linkage. Symbol ModifiedInternucleotidic Linkage s

s1

s2

s3

s4

s5

s6

s7

s8

s9

s10

s11

s12

s13

s14

s15

s16

s17

s18

For instance, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioateinternucleotidic linkage

between T and C; and 2) a phosphorothioate triester internucleotidiclinkage having the structure of

between C and G. Unless otherwise specified, the Rp/Sp designationspreceding an oligonucleotide sequence describe the configurations ofchiral linkage phosphorus atoms in the internucleotidic linkagessequentially from 5′ to 3′ of the oligonucleotide sequence. Forinstance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the “s” linkagebetween T and C has Rp configuration and the phosphorus in “s1” linkagebetween C and G has Sp configuration. In some embodiments, “All-(Rp)” or“All-(Sp)” is used to indicate that all chiral linkage phosphorus atomsin oligonucleotide have the same Rp or Sp configuration, respectively.For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ IDNO: 2) indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Rp configuration;All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 3)indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Sp configuration.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type”is used to define an oligonucleotide that has a particular basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).Oligonucleotides of a common designated “type” are structurallyidentical to one another.

One of skill in the art will appreciate that synthetic methods of thepresent disclosure provide for a degree of control during the synthesisof an oligonucleotide strand such that each nucleotide unit of theoligonucleotide strand can be designed and/or selected in advance tohave a particular stereochemistry at the linkage phosphorus and/or aparticular modification at the linkage phosphorus, and/or a particularbase, and/or a particular sugar. In some embodiments, an oligonucleotidestrand is designed and/or selected in advance to have a particularcombination of stereocenters at the linkage phosphorus. In someembodiments, an oligonucleotide strand is designed and/or determined tohave a particular combination of modifications at the linkagephosphorus. In some embodiments, an oligonucleotide strand is designedand/or selected to have a particular combination of bases. In someembodiments, an oligonucleotide strand is designed and/or selected tohave a particular combination of one or more of the above structuralcharacteristics. The present disclosure provides compositions comprisingor consisting of a plurality of oligonucleotide molecules (e.g.,chirally controlled oligonucleotide compositions). In some embodiments,all such molecules are of the same type (i.e., are structurallyidentical to one another). In many embodiments, however, providedcompositions comprise a plurality of oligonucleotides of differenttypes, typically in pre-determined relative amounts.

Chiral control: As used herein, “chiral control” refers to an ability tocontrol the stereochemical designation of every chiral linkagephosphorus within an oligonucleotide strand. The phrase “chirallycontrolled oligonucleotide” refers to an oligonucleotide which exists ina single diastereomeric form with respect to the chiral linkagephosphorus. Chirally controlled oligonucleotides are prepared fromchirally controlled oligonucleotide synthesis.

Chirally controlled oligonucleotide composition: As used herein, thephrase “chirally controlled oligonucleotide composition” refers to anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. For instance, in some embodiments achirally controlled oligonucleotide composition comprises oneoligonucleotide type. In some embodiments, a chirally controlledoligonucleotide composition comprises more than one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises a mixture of multiple oligonucleotide types.Example chirally controlled oligonucleotide compositions are describedfurther herein.

Chirally pure: as used herein, the phrase “chirally pure” is used todescribe a chirally controlled oligonucleotide composition in which allof the oligonucleotides exist in a single diastereomeric form withrespect to the linkage phosphorus.

Chirally uniform: as used herein, the phrase “chirally uniform” is usedto describe an oligonucleotide molecule or type in which all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, an oligonucleotide whose nucleotide units all have Rpstereochemistry at the linkage phosphorus is chirally uniform. Likewise,an oligonucleotide whose nucleotide units all have Sp stereochemistry atthe linkage phosphorus is chirally uniform.

Predetermined: By predetermined is meant deliberately selected, forexample as opposed to randomly occurring or achieved. Those of ordinaryskill in the art, reading the present specification, will appreciatethat the present disclosure provides new and surprising technologiesthat permit selection of particular oligonucleotide types forpreparation and/or inclusion in provided compositions, and furtherpermits controlled preparation of precisely the selected particulartypes, optionally in selected particular relative amounts, so thatprovided compositions are prepared. Such provided compositions are“predetermined” as described herein. Compositions that may containcertain individual oligonucleotide types because they happen to havebeen generated through a process that cannot be controlled tointentionally generate the particular oligonucleotide types is not a“predetermined” composition. In some embodiments, a predeterminedcomposition is one that can be intentionally reproduced (e.g., throughrepetition of a controlled process).

Linkage phosphorus: as defined herein, the phrase “linkage phosphorus”is used to indicate that the particular phosphorus atom being referredto is the phosphorus atom present in the internucleotidic linkage, whichphosphorus atom corresponds to the phosphorus atom of a phosphodiesterof an internucleotidic linkage as occurs in naturally occurring DNA andRNA. In some embodiments, a linkage phosphorus atom is in a modifiedinternucleotidic linkage, wherein each oxygen atom of a phosphodiesterlinkage is optionally and independently replaced by an organic orinorganic moiety. In some embodiments, a linkage phosphorus atom is P*of formula I. In some embodiments, a linkage phosphorus atom is chiral.In some embodiments, a chiral linkage phosphorus atom is P* of formulaI.

P-modification: as used herein, the term “P-modification” refers to anymodification at the linkage phosphorus other than a stereochemicalmodification. In some embodiments, a P-modification comprises addition,substitution, or removal of a pendant moiety covalently attached to alinkage phosphorus. In some embodiments, the “P-modification” is —X-L-R¹wherein each of X, L and R¹ is independently as defined and describedherein and below.

Blockmer: the term “blockmer,” as used herein, refers to anoligonucleotide strand whose pattern of structural featurescharacterizing each individual nucleotide unit is characterized by thepresence of at least two consecutive nucleotide units sharing a commonstructural feature at the internucleotidic phosphorus linkage. By commonstructural feature is meant common stereochemistry at the linkagephosphorus or a common modification at the linkage phosphorus. In someembodiments, the at least two consecutive nucleotide units sharing acommon structure feature at the internucleotidic phosphours linkage arereferred to as a “block”.

In some embodiments, a blockmer is a “stereoblockmer,” e.g., at leasttwo consecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. Such at least two consecutive nucleotide units forma “stereoblock.” For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmerbecause at least two consecutive nucleotide units, the Ts and the Cs1,have the same stereochemistry at the linkage phosphorus (both Sp). Inthe same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and itis a stereoblock.

In some embodiments, a blockmer is a “P-modification blockmer,” e.g., atleast two consecutive nucleotide units have the same modification at thelinkage phosphorus. Such at least two consecutive nucleotide units forma “P-modification block”. For instance, (Rp, Sp)-ATsCsGA is aP-modification blockmer because at least two consecutive nucleotideunits, the Ts and the Cs, have the same P-modification (i.e., both are aphosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

In some embodiments, a blockmer is a “linkage blockmer,” e.g., at leasttwo consecutive nucleotide units have identical stereochemistry andidentical modifications at the linkage phosphorus. At least twoconsecutive nucleotide units form a “linkage block”. For instance, (Rp,Rp)-ATsCsGA is a linkage blockmer because at least two consecutivenucleotide units, the Ts and the Cs, have the same stereochemistry (bothRp) and P-modification (both phosphorothioate). In the sameoligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is alinkage block.

In some embodiments, a blockmer comprises one or more blocksindependently selected from a stereoblock, a P-modification block and alinkage block. In some embodiments, a blockmer is a stereoblockmer withrespect to one block, and/or a P-modification blockmer with respect toanother block, and/or a linkage blockmer with respect to yet anotherblock. For instance, (Rp, Rp, Rp, Rp, Rp, Sp, Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG (SEQ ID NO: 4) is a stereoblockmer withrespect to the stereoblock AsTsCsGsAs1 (all Rp at linkage phosphorus) orTs1Cs1Gs1 (all Sp at linkage phosphorus), a P-modification blockmer withrespect to the P-modification block AsTsCsGs (all s linkage) orAs1Ts1Cs1Gs1 (all s1 linkage), or a linkage blockmer with respect to thelinkage block AsTsCsGs (all Rp at linkage phosphorus and all s linkage)or Ts1Cs1Gs1 (all Sp at linkage phosphorus and all s1 linkage).

Altmer: the term “altmer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is characterized in that no two consecutivenucleotide units of the oligonucleotide strand share a particularstructural feature at the internucleotidic phosphorus linkage. In someembodiments, an altmer is designed such that it comprises a repeatingpattern. In some embodiments, an altmer is designed such that it doesnot comprise a repeating pattern.

In some embodiments, an altmer is a “stereoaltmer,” e.g., no twoconsecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 5).

In some embodiments, an altmer is a “P-modification altmer” e.g., no twoconsecutive nucleotide units have the same modification at the linkagephosphorus. For instance, All-(Sp)-CAs1GsT, in which each linkagephosphorus has a different P-modification than the others.

In some embodiments, an altmer is a “linkage altmer,” e.g., no twoconsecutive nucleotide units have identical stereochemistry or identicalmodifications at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC (SEQ ID NO: 6).

Unimer: the term “unimer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is such that all nucleotide units within thestrand share at least one common structural feature at theinternucleotidic phosphorus linkage. By common structural feature ismeant common stereochemistry at the linkage phosphorus or a commonmodification at the linkage phosphorus.

In some embodiments, a unimer is a “stereounimer,” e.g., all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, All-(Sp)-CsAs1GsT, in which all the linkages have Spphosphorus.

In some embodiments, a unimer is a “P-modification unimer”, e.g., allnucleotide units have the same modification at the linkage phosphorus.For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO:7), in which all the internucleotidic linkages are phosphorothioatediester.

In some embodiments, a unimer is a “linkage unimer,” e.g., allnucleotide units have the same stereochemistry and the samemodifications at the linkage phosphorus. For instance,All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 8), inwhich all the internucleotidic linkages are phosphorothioate diesterhaving Sp linkage phosphorus.

Gapmer: as used herein, the term “gapmer” refers to an oligonucleotidestrand characterized in that at least one internucleotidic phosphoruslinkage of the oligonucleotide strand is a phosphate diester linkage,for example such as those found in naturally occurring DNA or RNA. Insome embodiments, more than one internucleotidic phosphorus linkage ofthe oligonucleotide strand is a phosphate diester linkage such as thosefound in naturally occurring DNA or RNA. For instance, All-(Sp)-CAs1GsT,in which the internucleotidic linkage between C and A is a phosphatediester linkage.

Skipmer: as used herein, the term “skipmer” refers to a type of gapmerin which every other internucleotidic phosphorus linkage of theoligonucleotide strand is a phosphate diester linkage, for example suchas those found in naturally occurring DNA or RNA, and every otherinternucleotidic phosphorus linkage of the oligonucleotide strand is amodified internucleotidic linkage. For instance,All-(Sp)-AsTCs1GAs2TCs3G.

For purposes of this disclosure, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The methods and structures described herein relating to compounds andcompositions of the disclosure also apply to the pharmaceuticallyacceptable acid or base addition salts and all stereoisomeric forms ofthese compounds and compositions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Reverse phase HPLCs after incubation with rat liver homogenate.Total amounts of oligonucleotides remaining when incubated with ratwhole liver homogenate at 37° C. at different days were measured. Thein-vitro metabolic stability of ONT-154 was found to be similar toONT-87, which has 2′-MOE wings, while both have much better stabilitythan 2′-MOE gapmer which is stereorandom (ONT-41, Mipomersen). Theamount of full length oligomer remaining was measured by reverse phaseHPLC where peak area of the peak of interest was normalized withinternal standard.

FIG. 2. Degradation of various chirally pure analogues of Mipomersen(ONT-41) in rat whole liver homogenate. Total amounts of oligonucleotideremaining when incubated with rat whole liver homogenate at 37° C. atdifferent days were measured. The in-vitro metabolic stability ofchirally pure diastereomers of human ApoB sequence ONT-41 (Mipomersen)was found to increase with increased Sp internucleotidic linkages. Theamount of full length oligomer remaining was measured by reverse phaseHPLC where peak area of the peak of interest was normalized withinternal standard. Compositions used include: ONT-41, ONT-75, ONT-77,ONT-80, ONT-81, ONT-87, ONT-88 and ONT-89.

FIG. 3. Degradation of various chirally pure analogues of mouse ApoBsequence (ISIS 147764, ONT-83) in rat whole liver homogenate. Totalamounts of oligonucleotide remaining when incubated with rat whole liverhomogenate at 37° C. at different days were measured. The in-vitrometabolic stability of chirally pure diastereomers of murine ApoBsequence (ONT-83, 2′-MOE gapmer, stereorandom phosphorothioate) wasfound to increase with increased Sp internucleotidic linkages. Theamount of full length oligomer remaining was measured by reverse phaseHPLC where peak area of the peak of interest was normalized withinternal standard. Compositions used include: ONT-82 to ONT-86.

FIG. 4. Degradation of Mipomersen analogue ONT-75 in rat whole liverhomogenate over a period of 24 hrs. This figure illustrates stability ofONT-75 in rate whole liver homogenate.

FIG. 5. Degradation of Mipomersen analogue ONT-81 in rat whole liverhomogenate over a period of 24 hrs. This figure illustrates stability ofONT-81 in rate whole liver homogenate.

FIG. 6. Durations of knockdown for ONT-87, ONT-88, and ONT-89.Stereoisomers can exhibit substantially different durations ofknockdown. ONT-87 results in substantially more durable suppression thanother stereoisomers. Increased duration of action of ONT-87 in multiplein vivo studies was observed. ONT-88 showed similar efficacy andrecovery profile as ONT-41 (Mipomersen) in certain in-vivo studies. HuApoB transgenic mice, n=4, were dosed with 10 mpk IP bolus, 2×/week forthree weeks. The mice were randomized to study groups, and dosedintraperitoneally (IP) at 10 mg/kg on Days 1, 4, 8, 11, 15, 18, and 22,based on individual mouse body weight measured prior to dosing on eachdosing day. Blood was collected on days 0, 17, 24, 31, 38, 45 and 52 bysubmandibular (cheek) bleed and at sacrifice on Day 52 by cardiacpuncture and then processed to serum. ApoB was measured by ELISA.Highlighted: 72% vs. 35% knock-down maintained at 3 weeks postdose.

FIG. 7. HPLC profiles exhibiting the difference in metabolic stabilitydetermined in Human Serum for siRNA duplexes having several Rp, Sp orstereorandom phosphorothioate linkages. Compositions used include:ONT-114, ONT-116, ONT-109, ONT-107, ONT-108 and ONT-106.

FIG. 8. Effect of stereochemistry on RNase H activity. Oligonucleotideswere hybridized with RNA and then incubated with RNase H at 37° C. inthe presence of 1×RNase H buffer. From top to bottom at 120 min: ONT-89,ONT-77, ONT-81, ONT-80, ONT-75, ONT-41, ONT-88, ONT-154, ONT-87, withONT-77/154 very close to each other.

FIG. 9. Analysis of human RNase H1 cleavage of a 20-mer RNA whenhybridized with different preparations of stereoisomers ofphosphorothioate oligonucleotides targeting the same region of humanApoB mRNA. Specific sites of cleavage are strongly influenced by thedistinct stereochemistries. Arrows represent position of cleavage(cleavage sites). Products were analyzed by UPLC/MS. The length of thearrow signifies the amount of products present in the reaction mixturewhich was determined from the ratio of UV peak area to theoreticalextinction coefficient of that fragment (the larger the arrow, the morethe detected cleavage products). (A): Legend for cleavage maps. (B) and(C): cleavage maps of oligonucleotides. In the figures: (τ) indicatesthat both RNase H1 cleavage fragments (5′-phosphate species as well as5′-OH 3′-OH species) were identified in reaction mixtures. (┌) indicatesthat only 5′-phosphate species was detected and (┐) indicates that 5′-OH3′-OH component was detected in mass spectrometry analysis. Compositionsused include: ONT-41, ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88,ONT-89 and ONT-154 (SEQ ID NOS 397-401 and 758-761, respectively).Figure also discloses SEQ ID NO: 1559.

FIG. 10. Cleavage maps of different oligonucleotide compositions((A)-(C)). These three sequences target different regions in FOXO1 mRNA.Each sequence was studied with five different chemistries. Cleavage mapsare derived from reaction mixtures obtained after 30 minutes ofincubation of respective duplexes with RNase H1C in the presence of1×PBS buffer at 37° C. Arrows indicate sites of cleavage. The length ofthe arrow signifies the amount of products present in the reactionmixture which was determined from the ratio of UV peak area totheoretical extinction coefficient of that fragment (the larger thearrow, the more the detectable cleavage products). Only in the caseswhere 5′-OH 3′-OH was not detected in the reaction mixture, 5′-phosphatespecies peak was used for quantification. Cleavage rates were determinedby measuring amount of full length RNA remaining in the reactionmixtures by reverse phase HPLC. Reactions were quenched at fixed timepoints by 30 mM Na₂EDTA. Compositions used include: ONT-316, ONT-355,ONT-361, ONT-367, ONT-373, ONT-302, ONT-352, ONT-358, ONT-364, ONT-370,ONT-315, ONT-354, ONT-360, ONT-366, and ONT-372 (SEQ ID NOS 670-674,676-680 and 682-686, respectively). Figure also discloses SEQ ID NO:1560 in panel A and SEQ ID NO: 1561 in panels B and C.

FIG. 11. Cleavage maps of oligonucleotide compositions having differentcommon base sequences and lengths ((A)-(B)). The maps show a comparisonof stereorandom DNA compositions (top panel) with three distinct andstereochemically pure oligonucleotide compositions. Data compare resultsof chirally controlled oligonucleotide compositions with twostereorandom phosphorothioate oligonucleotide compositions (ONT-366 andONT-367) targeting different regions in FOXO1 mRNA. Each panel shows acomparison of stereorandom DNA (top panel) with three distinct andstereochemically pure oligonucleotide preparations. Cleavage maps werederived from reaction mixtures obtained after 30 minutes of incubationof respective duplexes with RNase H1C in the presence of 1×PBS buffer at37° C. Arrows indicate sites of cleavage. The length of the arrowsignifies the amount of metabolite present in the reaction mixture whichwas determined from the ratio of UV peak area to theoretical extinctioncoefficient of that fragment (the larger the arrow, the more thedetectable cleavage products). Only in the cases where 5′-OH 3′-OH wasnot detected in the reaction mixture, 5′-phosphate species peak was usedfor quantification. Compositions used include: ONT-366, ONT-389,ONT-390, ONT-391, ONT-367, ONT-392, ONT-393, and ONT-394 (SEQ ID NOS660-663 and 665-668, respectively). Figure also discloses SEQ ID NO:1562 in panel A and SEQ ID NO: 1560 in panel B.

FIG. 12. Effect of stereochemistry on RNase H activity. In twoindependent experiments, antisense oligonucleotides targeting anidentical region of FOXO1 mRNA were hybridized with RNA and thenincubated with RNase H at 37° C. in the presence of 1×RNase H buffer.Disappearance of full length RNA was measured from its peak area at 254nm using RP-HPLC. (A): from top to bottom at 60 min: ONT-355, ONT-316,ONT-367, ONT-392, ONT-393 and ONT-394 (ONT-393 and ONT-394 about thesame at 60 min; ONT-393 had higher % RNA substrate remaining at 5 min).(B): from top to bottom at 60 min: ONT-315, ONT-354, ONT-366, ONT-391,ONT-389 and ONT-390. Cleavage rates were determined by measuring amountof full length RNA remaining in the reaction mixtures by reverse phaseHPLC. Reactions were quenched at fixed time points by 30 mM Na₂EDTA.

FIG. 13. Turnover of antisense oligonucleotides. The duplexes were madewith each DNA strand concentration equal to 6 μM and RNA being 100 μM.These duplexes were incubated with 0.02 μM RNase H enzyme anddisappearance of full length RNA was measured from its peak area at 254nm using RP-HPLC. Cleavage rates were determined by measuring amount offull length RNA remaining in the reaction mixtures by reverse phaseHPLC. Reactions were quenched at fixed time points by 30 mM Na₂EDTA.From top to bottom at 40 min: ONT-316, ONT-367 and ONT-392.

FIG. 14. Cleavage map comparing a stereorandom phosphorothioateoligonucleotide with six distinct and stereochemically pureoligonucleotide preparations targeting the same FOXO1 mRNA region.Compositions used include: ONT-367, ONT-392, ONT-393, ONT-394, ONT-400,ONT-401, and ONT-406 (SEQ ID NOS 665-668, 729-730 and 735,respectively). Figure also discloses SEQ ID NO: 1560.

FIG. 15. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides were hybridized with RNA and then incubated with RNaseH at 37° C. in the presence of 1×RNase H buffer. Dependence ofstereochemistry upon RNase H activity was observed. Also evident incomparing ONT-367 (stereorandom DNA) and ONT-316 (5-10-5 2′-MOE Gapmer)is the strong dependence of compositional chemistry upon RNase Hactivity. From top to bottom at 40 min: ONT-316, ONT-421, ONT-367,ONT-392, ONT-394, ONT-415, and ONT-422 (ONT-394/415/422 have similarlevels at 40 min; at 5 min, ONT-422>ONT-394>ONT-415 in % RNA remainingin DNA/RNA duplex).

FIG. 16. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides targeting an identical region of FOXO1 mRNA werehybridized with RNA and then incubated with RNase H at 37° C. in thepresence of 1×RNase H buffer. Dependence of stereochemistry upon RNase Hactivity was observed. Form top to bottom at 40 min: ONT-396, ONT-409,ONT-414, ONT-408 (ONT-396/409/414/408 have similar levels at 40 min),ONT-404, ONT-410, ONT-402 (ONT-404/410/408 have similar levels at 40min), ONT-403, ONT-407, ONT-405, ONT-401, ONT-406 and ONT-400(ONT-401/405/406/400 have similar levels at 40 min).

FIG. 17. Effect of stereochemistry on RNase H activity. Antisenseoligonucleotides targeting an identical region of FOXO1 mRNA werehybridized with RNA and then incubated with RNase H at 37° C. in thepresence of 1×RNase H buffer. Dependence of stereochemistry upon RNase Hactivity was observed. ONT-406 was observed to elicit cleavage ofduplexed RNA at a rate in slight excess of that of the phosphodiesteroligonucleotide ONT-415. From top to bottom at 40 min: ONT-396, ONT-421,ONT-392, ONT-394, ONT-415 ONT-406, and ONT-422 (ONT-394/415/406 havesimilar levels at 40 min; at 5 min, ONT-394>ONT-415>ONT-406 in % RNAremaining in DNA/RNA duplex).

FIG. 18. Example UV chromatograms of RNA cleavage products obtained whenRNA (ONT-388) was duplexed with stereorandom DNA, ONT-367 (top) andstereopure DNA with repeat triplet motif-3′-SSR-5′, ONT-394 (bottom). ).2.35 min: 7mer; 3.16 min: 8mer and p-6mer; 4.48 min: P-7mer; 5.83 min:P-8mer; 6.88 min: 12mer; 9.32 min: 13mer; 10.13 min: P-11mer; 11.0 min:P-12mer and 14mer; 11.93 min: P-13mer; 13.13 min: P-14mer. ONT-394 (onthe bottom) peak assignment: 4.55 min: p-7mer; 4.97 min: 10mer; 9.53min: 13mer.

FIG. 19. Electrospray Ionization Spectrum of RNA cleavage products. RNAfragments obtained from the duplex ONT-387, RNA/ONT-354, (7-6-7,DNA-2′-OMe-DNA) on the top and ONT-387, RNA/ONT-315, (5-10-5,2′-MOEGapmer) at the bottom when these duplexes were incubated with RNase Hfor 30 min in the presence of 1×RNase H buffer.

FIG. 20. UV Chromatogram and TIC of ONT-406 and ONT-388 duplex after 30minutes of incubation with RNase H.

FIG. 21. An example proposed cleavage. Provided chirally controlledoligonucleotide compositions are capable of cleaving targets asdepicted.

FIG. 22. Example allele specific cleavage targeting mutant HuntingtinmRNA. (A) and (B): example oligonucleotides. (C)-(E): cleavage maps.(F)-(H): RNA cleavage. Stereorandom and chirally controlledoligonucleotide compositions were prepared to target single nucleotidepolymorphisms for allele selective suppression of mutant Huntingtin.ONT-450 (stereorandom) targeting ONT-453 (muHTT) and ONT-454 (wtHTT)showed marginal differentiation in RNA cleavage and their cleavage maps.Chirally controlled ONT-451 with selective placement of 3′-SSR-5′ motifin RNase H recognition site targeting ONT-453 (muHTT) and ONT-454(wtHTT) showed large differentiation in RNA cleavage rate. From thecleavage map, it is notable that 3′-SSR-5′ motif is placed to direct thecleavage between positions 8 and 9 which is after the mismatch if readfrom 5′-end of RNA. ONT-452 with selective placement of 3′-SSR-5′ motifin RNase H recognition site targeting ONT-453 (muHTT) and ONT-454(wtHTT) showed moderate differentiation in RNA cleavage rate. 3′-SSR-5′motif was placed to direct the cleavage at positions 7 and 8 which isbefore the mismatch if read from 5′-end of RNA. Example data illustratesignificance of position in placement of 3′-SSR-5′ motif to achieveenhanced discrimination for allele specific cleavage. All cleavage mapsare derived from the reaction mixtures obtained after 5 minutes ofincubation of respective duplexes with RNase H1C in the presence of1×PBS buffer at 37° C. Arrows indicate sites of cleavage. The length ofthe arrow signifies the amount of metabolite present in the reactionmixture which was determined from the ratio of UV peak area totheoretical extinction coefficient of that fragment. Only in the caseswhere 5′-OH 3′-OH was not detected in the reaction mixture, 5′-phosphatespecies peak was used for quantification. Compositions used include:ONT-450 to ONT-454 (SEQ ID NOS 380, 267, 268, 799 and 718,respectively). Figure also discloses SEQ ID NOS 1563-1565 in panels C-E,respectively, in order of appearance.

FIG. 23. (A)-(C): example allele specific cleavage targeting FOXO1 mRNA(SEQ ID NOS 669, 731, 715, 731, 699, 729, 715, 729, 699, 735, 716 and735, respectively, in order of appearance).

FIG. 24. In vitro dose response silencing of ApoB mRNA after treatmentwith ApoB oligonucleotides. Stereochemically pure diastereomers with andwithout 2′-MOE wings show similar efficacy as ONT-41 (Mipomersen).Compositions used include: ONT-87, ONT-41, and ONT-154.

FIG. 25. Comparison of RNase H cleavage maps (A) and RNA cleavage rates(B) for stereorandom composition (ONT-367 (SEQ ID NO: 769)) and chirallycontrolled oligonucleotide compositions (ONT-421 (SEQ ID NO: 772), allSp and ONT-455 (SEQ ID NO: 767), all Rp) and DNA (ONT-415 (SEQ ID NO:770)). These sequences target the same region in FOXO1 mRNA. Cleavagemaps were derived from the reaction mixtures obtained after 5 minutes ofincubation of respective duplexes with RNase H1C in the presence of1×PBS buffer at 37° C. Arrows indicate sites of cleavage. The length ofthe arrow signifies the amount of metabolite present in the reactionmixture which was determined from the ratio of UV peak area totheoretical extinction coefficient of that fragment. Only in the caseswhere 5′-OH 3′-OH was not detected in the reaction mixture, 5′-phosphatespecies peak was used for quantification. Cleavage rates were determinedby measuring amount of full length RNA remaining in the reactionmixtures by reverse phase HPLC. Reactions are quenched at fixed timepoints by 30 mM Na₂EDTA. Figure also discloses SEQ ID NO: 1560.

FIG. 26. Comparison of cleavage maps of sequences containing one Rp withchange of position starting from 3′-end of DNA. Compositions usedinclude: ONT-396 to ONT-414 (SEQ ID NOS 725-743, respectively). Thesesequences target the same region in FOXO1 mRNA. Cleavage maps arederived from the reaction mixtures obtained after 5 minutes ofincubation of respective duplexes with RNase H1C in the presence of1×RNase H buffer at 37° C. Arrows indicate sites of cleavage. The lengthof the arrow signifies the amount of metabolite present in the reactionmixture which was determined from the ratio of UV peak area totheoretical extinction coefficient of that fragment. Only in the caseswhere 5′-OH 3′-OH was not detected in the reaction mixture, 5′-phosphatespecies peak was used for quantification. Figure also discloses SEQ IDNO: 1560 in panels A-D.

FIG. 27. (A) Comparison of RNase H cleavage rates for stereopureoligonucleotides (ONT-406), (ONT-401), (ONT-404) and (ONT-408). All foursequences are stereopure phosphorothioates with one Rp linkage. Thesesequences target the same region in FOXO1 mRNA. All duplexes wereincubation with RNase H1C in the presence of 1×RNase H buffer at 37° C.Reactions were quenched at fixed time points by 30 mM Na₂EDTA. Cleavagerates were determined by measuring amount of full length RNA remainingin the reaction mixtures by reverse phase HPLC. ONT-406 and ONT-401 werefound to have superior cleavage rates. (B) Correlation between % RNAcleaved in RNase H assay (10 μM oligonucleotide) and % mRNA knockdown inin vitro assay (20 nM oligonucleotide). All sequences target the sameregion of mRNA in the FOXO1 target. The quantity of RNA remaining isdetermined by UV peak area for RNA when normalized to DNA in the samereaction mixture. All of the above maps are derived from the reactionmixture obtained after 5 minutes of incubation of respective duplexeswith RNase H1C in the presence of 1×PBS buffer at 37° C. All sequencesfrom ONT-396 to ONT-414 have one Rp phosphorothioate and they vary inthe position of Rp. ONT-421 (All Sp) phosphorothioate was inactivein-vitro assay. It relates poor cleavage rate of RNA in RNase H assaywhen ONT-421 is duplexed with complementary RNA.

FIG. 28. Serum stability assay of single Rp walk PS DNA(ONT-396-ONT-414), stereorandom PS DNA(ONT-367), all-Sp PS DNA (ONT-421)and all-Rp PS DNA (ONT-455) in rat serum for 2 days. Note ONT-396 andONT-455 decomposed at tested time point. Compositions used include:ONT-396 to ONT-414, ONT-367, ONT-421, and ONT-455.

FIG. 29. Example oligonucleotides including hemimers. (A): cleavagemaps. (B): RNA cleavage assay. (C): FOXO1 mRNA knockdown. ONT-440 (SEQID NO: 765), ONT-441 (SEQ ID NO: 766), and ONT-367 (SEQ ID NO: 769) areused. In some embodiments, introduction of 2′-modifications on 5′-end ofthe sequences increases stability for binding to target RNA whilemaintaining RNase H activity. ONT-367 (stereorandom phosphorothioateDNA) and ONT-440 (5-15, 2′-F-DNA) have similar cleavage maps and similarrate of RNA cleavage in RNase H assay (10 LM oligonucleotide). In someembodiments, ONT-440 (5-11, 2′-F-DNA) sequence can have better cellpenetration properties. In some embodiments, asymmetric 2′-modificationsprovide Tm advantage while maintaining RNase H activity. Introduction ofRSS motifs can further enhance RNase H efficiency in the hemimers.Cleavage maps are derived from the reaction mixtures obtained after 5minutes of incubation of respective duplexes with RNase H1C in thepresence of 1×RNase H buffer at 37° C. Arrows indicate sites ofcleavage. (τ) indicates that both fragments, 5′-phosphate species aswell as 5′-OH 3′-OH species were identified in reaction mixtures. (┌)indicates that only 5′-phosphate species was detected and (┐) indicatesthat 5′-OH 3′-OH component was detected in mass spectrometry analysis.The length of the arrow signifies the amount of metabolite present inthe reaction mixture which was determined from the ratio of UV peak areato theoretical extinction coefficient of that fragment. Only in thecases where 5′-OH 3′-OH was not detected in the reaction mixture,5′-phosphate species peak was used for quantification. Figure alsodiscloses SEQ ID NO: 1560 in panel A.

FIG. 30. Example mass spectrometry data of cleavage assay. Top: data forONT-367: 2.35 min: 7 mer; 3.16 min: 8 mer and P-6 mer; 4.58 min: P-7mer; 5.91 min: P-8 mer; 7.19 min: 12 mer; 9.55 min: 13 mer; 10.13 min:P-11 mer; 11.14 min: P-12 mer and 14 mer; 12.11 min: P-13 mer; 13.29min: P-14 mer; 14.80 min: full length RNA (ONT-388) and 18.33 min:stereorandom DNA (ONT-367). Bottom: data for ONT-406: 4.72 min:p-rArUrGrGrCrUrA, 5′-phosphorylated 7 mer RNA; 9.46 min:5′-rGrUrGrArGrCrArGrCrUrGrCrA (SEQ ID NO: 9), 5′-OH 3′-OH 13 mer RNA;16.45 min: full length RNA (ONT-388); 19.48 and 19.49 min: stereopureDNA (ONT-406).

FIG. 31. Example RNA cleavage rates. Duplexes were incubated with RNaseH1C in the presence of 1×RNase H buffer at 37° C. Reactions werequenched at fixed time points by addition of 30 mM Na₂EDTA. Cleavagerates were determined by measuring amount of full length RNA remainingin the reaction mixtures. Compositions used include: WV-944 (SEQ ID NO:791), WV-945 (SEQ ID NO: 792), WV-936 (SEQ ID NO: 162), WV-904 (SEQ IDNO: 130), WV-937 (SEQ ID NO: 163), WV-905 (SEQ ID NO: 131), WV-938 (SEQID NO: 164), WV-906 (SEQ ID NO: 132), WV-939 (SEQ ID NO: 165), WV-907(SEQ ID NO: 133), WV-940 (SEQ ID NO: 166), WV-908 (SEQ ID NO: 34),WV-941 (SEQ ID NO: 167), and WV-909 (SEQ ID NO: 135).

FIG. 32. A-N: RNA cleavage rates in RNase H assay for certaincompositions targeting rs362307. Some of these compositions arestereorandom and some chirally controlled. Compositions used include:WV-1085, WV-1086, WV-1087, WV-1088, WV-1089, WV-1090, WV-1091, WV-1092,WV-905, WV-944, WV-945, WV-911, WV-917, WV-931, WV-937, and WV-1497.

FIG. 33. A: Example cleavage maps. Cleavage maps were derived fromreaction mixtures obtained after 5 minutes of incubation of respectiveduplexes with RNase H1C in the presence of 1×RNaseH buffer at 37° C. B:Legend. Arrows indicate sites of cleavage. (τ) indicates that bothfragments, 5′-phosphate species as well as 3′-OH species wereidentified. (┌) indicates that only 5′-OH 3′-OH species was detected and(┐) indicates that 5′-Phosphate component was detected. Length of anarrow signifies the amount of fragment present in the reaction mixturewhich was determined from the ratio of UV peak area to theoreticalextinction coefficient of that fragment. Only in the cases where 5′-OH3′-OH fragments were not detected in the reaction mixture, the5′-phosphate species peak was used for quantification. Compositions usedinclude: WV-944 (SEQ ID NO: 791), WV-945 (SEQ ID NO: 792), WV-904 (SEQID NO: 130), WV-905 (SEQ ID NO: 131), WV-906 (SEQ ID NO: 132), WV-907(SEQ ID NO: 133), WV-908 (SEQ ID NO: 134), and WV-909 (SEQ ID NO: 135).

FIG. 34. Example cleavage maps. Example cleavage maps. Cleavage mapswere derived from reaction mixtures obtained after 30 minutes ofincubation of respective duplexes with RNase H1C in the presence of1×RNase H buffer at 37° C. For legend, see FIG. 33. Compositions usedinclude: WV-944 (SEQ ID NO: 791), WV-945 (SEQ ID NO: 792), WV-936 (SEQID NO: 162), WV-937 (SEQ ID NO: 163), WV-938 (SEQ ID NO: 164), WV-939(SEQ ID NO: 165), WV-940 (SEQ ID NO: 166), WV-941 (SEQ ID NO: 167),WV-1085 (SEQ ID NO: 168), WV-1086 (SEQ ID NO: 169), WV-1087 (SEQ ID NO:170), WV-1088 (SEQ ID NO: 171), WV-1089 (SEQ ID NO: 172), WV-1090 (SEQID NO: 173), WV-1091 (SEQ ID NO: 174), and WV-1092 (SEQ ID NO: 175).

FIG. 35. Example cleavage maps. For legend, see FIG. 33. Compositionsused include: WV-944 (SEQ ID NO: 791), WV-945 (SEQ ID NO: 792), WV-905(SEQ ID NO: 131), WV-911 (SEQ ID NO: 137), WV-917 (SEQ ID NO: 143),WV-931 (SEQ ID NO: 157), and WV-937 (SEQ ID NO: 163).

FIG. 36. Total ion chromatogram of RNase H cleavage reaction for WV-937when duplexed with WT HTT RNA (WV-944, upper panel) or mu HTT RNA(WV-945, lower panel). Following quenching of the enzymatic reactionwith disodium EDTA after 30 minutes, the RNase H cleavage products werechromatographically resolved and analyzed using an Agilent 1290 UPLCcoupled with an Agilent 6230 MS-TOF mass spectrometer. The high massaccuracy high resolution MS spectra for each identified peak wasextracted and deconvoluted. Identification of the metabolites which ledto determination of position of cleavage was done by comparing thedeconvoluted average masses to masses of predicted RNA metabolites.

FIG. 37. Illustration of the Luciferase Reporter-based screening.

FIGS. 38A-38I. FIGS. 38A-38I and 39A-39G show the activity of variousHTT oligonucleotides. Dose-response curves for HTT silencing inreporter-based assay in COS7 cells after transfection of ASOs targetingrs362331_T or rs2530595_T SNPs. ASO specificity is increased with nosignificant loss of potency by addition of stereopure design (calculatedIC50s specified). Data are representative of 2 independent experiments.Lines indicate fit curves, error bars indicate standard deviations. Inthe figures, the location of the SNP is indicated. Compositions testedin FIG. 38 include: WV-2067, WV-2416, WV-2069, WV-2417, WV-2072,WV-2418, WV-2076, WV-2419, WV-2605, WV-2589, WV-2606, WV-2590, WV-2607,WV-2591, WV-2608, WV-2592, WV-2609, WV-2593, WV-2610, WV-2594, WV-2611,WV-2595, WV-2612, WV-2596, WV-2611, WV-2595, WV-2671, WV-2672, WV-2673,WV-2675, WV-2674, WV-2613, WV-2597, WV-2614, WV-2598, WV-2615, WV-2599,WV-2616, WV-2600, WV-2617, WV-2601, WV-2618, WV-2602, WV-2619, WV-2603,WV-2620, and WV-2604. FIG. 38F discloses SEQ ID NOS 1475, 1459 and1487-1491, respectively, in order of appearance.

FIGS. 39A-39G. Dose-response curves for HTT silencing in reporter-basedassay in COS7 cells after transfection of ASOs. Compositions testedinclude: WVE120101, WV-1092, WV-1497, WV-2619, WV-2603, WV-2611, andWV-2595. IC₅₀ data is also shown. FIG. 39A discloses SEQ ID NOS 1483 and1467, respectively, in order of appearance. FIG. 39B discloses SEQ IDNOS 1483 and 1467, respectively, in order of appearance. FIG. 39Cdiscloses SEQ ID NOS 1483 and 1467, respectively, in order ofappearance. FIG. 39E discloses SEQ ID NOS 1483 and 1467, respectively,in order of appearance. FIG. 39F discloses SEQ ID NOS 1475 and 1459,respectively, in order of appearance. FIG. 39G discloses SEQ ID NOS 1483and 1467, respectively, in order of appearance.

FIGS. 40A-40D. FIGS. 40A-40D shows liquid chromatograph and mass spectradata for oligonucleotides: WV1092.22 (WV-1092), WV2595.01 (WV-2595) andWV2603.01 (WV-2603). The suffices (01), (02), 0.01, 0.02, 0.22, etc., asused herein, indicate batch numbers.

FIG. 41. FIG. 41 shows liquid chromatograph and mass spectra data foroligonucleotides: WV-1510, WV-2378 and WV-2380.

FIG. 42. Example tested sequences (SEQ ID NOS 775-782, respectively, inorder of appearance).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Synthetic oligonucleotides provide useful molecular tools in a widevariety of applications. For example, oligonucleotides are useful intherapeutic, diagnostic, research, and new nanomaterials applications.The use of naturally occurring nucleic acids (e.g., unmodified DNA orRNA) is limited, for example, by their susceptibility to endo- andexo-nucleases. As such, various synthetic counterparts have beendeveloped to circumvent these shortcomings. These include syntheticoligonucleotides that contain backbone modifications, which render thesemolecules less susceptible to degradation. From a structural point ofview, such modifications to internucleotide phosphate linkages introducechirality. It has become clear that certain properties ofoligonucleotides may be affected by the configurations of the phosphorusatoms that form the backbone of the oligonucleotides. For example, invitro studies have shown that the properties of antisense nucleotidessuch as binding affinity, sequence specific binding to the complementaryRNA, stability to nucleases are affected by, inter alia, chirality ofthe backbone (e.g., the configurations of the phosphorus atoms).

Among other things, the present disclosure encompasses the recognitionthat structural elements of oligonucleotides, such as base sequence,chemical modifications (e.g., modifications of sugar, base, and/orinternucleotidic linkages, and patterns thereof), and/or stereochemistry(e.g., stereochemistry of backbone chiral centers (chiralinternucleotidic linkages), and/or patterns thereof), can havesignificant impact on properties, e.g., activities, of oligonucleotides.In some embodiments, the present disclosure demonstrates thatoligonucleotide compositions comprising oligonucleotides with controlledstructural elements, e.g., controlled chemical modification and/orcontrolled backbone stereochemistry patterns, provide unexpectedproperties, including but not limited to those described herein. In someembodiments, the present disclosure provide an oligonucleotidecomposition comprises a predetermined level of oligonucleotides of anindividual oligonucleotide type which are chemically identical, e.g.,they have the same base sequence, the same pattern of nucleosidemodifications (modifications to sugar and base moieties, if any), thesame pattern of backbone chiral centers, and the same pattern ofbackbone phosphorus modifications.

Among other things, the present disclosure encompasses the recognitionthat stereorandom oligonucleotide preparations contain a plurality ofdistinct chemical entities that differ from one another, e.g., in thestereochemical structure of individual backbone chiral centers withinthe oligonucleotide chain. Without control of stereochemistry ofbackbone chiral centers, stereorandom oligonucleotide preparationsprovide uncontrolled compositions comprising undetermined levels ofoligonucleotide stereoisomers. Even though these stereoisomers may havethe same base sequence, they are different chemical entities at leastdue to their different backbone stereochemistry, and they can have, asdemonstrated herein, different properties, e.g., bioactivities. Astereopure (or “chirally controlled”) oligonucleotide composition orpreparation can have improved bioactivity compared to a stereorandomoligonucleotide preparation which is otherwise identical (e.g., both thestereopure and stereorandom versions have the same base sequence,pattern of base and sugar modifications, etc.). For example,stereorandom oligonucleotide WV-1497 composition and a stereopureoligonucleotide WV-1092 composition both have the same sequence of basesand identical patterns of sugar modifications and backbone linkages,differing only in stereochemistry. However, at higher concentrations,there was a marked difference in the ability of the stereopure WV-1092composition and the stereorandom WV-1497 composition to differentiatebetween wt and mutant HTT (which differ in only one nt). At the highconcentration, both knocked down the mutant HTT to a great degree, whichis desirable; but stereopure WV-1092 showed only a small knock down ofwildtype HTT, while WV-1497 showed significantly more knock down of wtHTT, which is less desirable in some instances.

Chirally controlled oligonucleotide compositions of both WVE120101 andWV-1092 were able to differentiate between wt and mutant versions of SNPrs362307, which differ by one nt; both WVE120101 and WV-1092significantly knocked down the mutant allele but not the wt, while thestereorandom version, WV-1497, was not able to significantlydifferentiate between the wt and mutant alleles (see FIG. 39D). Themodified sequences of WVE120101 and WV-1092 are identical.

A chirally controlled oligonucleotide composition of WV-2595 was able todifferentiate between the C and T alleles at SNP rs2530595, which alsodiffer at only the one nt. Stereopure WV-2595 significantly knocked downthe T allele but not the C allele, unlike the stereorandomoligonucleotide composition of WV-2611, which was not able tosignificantly differentiate the alleles (see FIG. 39F). The sequence ofWV-2595 is 5′-mG*mGmGmUmCC*T*C*C*C*C*A*C*A*G*mAmGmGmG*mA-3′ (SEQ ID NO:10) or 5′-mG* SmGmGmUmC*SC*ST*SC* SC*SC*SC*SA*SC*RA* SG*SmAmGmGmG*SmA-3′ (SEQ ID NO: 11) with certain stereochemistry information.

A stereopure oligonucleotide composition of WV-2603 was able todifferentiate between the C and T alleles of SNP rs362331, which alsodiffer at only the one nt. Stereopure WV-2603 significantly knocked downthe T allele but not the C allele, unlike the stereorandomoligonucleotide composition of WV-2619, which was not able tosignificantly differentiate between the alleles (see FIGS. 39A, 39B, 39Cand 39E). The sequence of WV-2603 is5′-mG*mUmGmCmA*C*A*C*A*G*T*A*G*A*T*mGmAmGmG*mG-3′ (SEQ ID NO: 12) or5′-mG* SmUmGmCmA*SC*SA*SC* SA*SG*ST*SA*SG*RA* ST*SmGmAmGmG* SmG-3′ (SEQID NO: 13) with certain stereochemistry information.

In some embodiments, the sequence of the oligonucleotide in a stereopure(chirally controlled) oligonucleotide composition comprises or consistsof the sequence of any oligonucleotide disclosed herein. In someembodiments, the sequence of the oligonucleotide in a stereopure(chirally controlled) oligonucleotide composition comprises or consistsof the sequence of any oligonucleotide selected from Tables N1, N2, N3,N4 and 8. In some embodiments, the sequence of the oligonucleotide in astereopure (chirally controlled) oligonucleotide composition comprisesor consists of the sequence of any oligonucleotide selected from TablesN1A, N2A, N3A, N4A and 8. In some embodiments, the sequence of theoligonucleotide in a stereopure (chirally controlled) oligonucleotidecomposition comprises or consists of the sequence of WV-1092, WVE120101,WV-2603 or WV-2595.

Each oligonucleotide described herein comprising a HTT sequencerepresents an HTT oligonucleotide which was designed, constructed andtested in various assays, in some embodiments, one or more in vitroassays. Each HTT oligonucleotide listed in any of Tables N1A, N2A, N3A,N4A and 8, or described elsewhere herein, was designed, constructed andtested in various assays, in some embodiments, one or more in vitroassays. For example, HTT oligonucleotides described herein were testedin a dual luciferase reporter assay. In some embodiments, HTToligonucleotides were tested in one or more other assays described inthis disclosure and/or in the art in accordance with the presentdisclosure. In some embodiments, HTT oligonucleotides which were foundto be particularly efficacious in the dual luciferase assay were testedin further in vitro and in vivo assays in accordance with the presentdisclosure.

In some embodiments, a sequence of an oligonucleotide in a stereopure(chirally controlled) oligonucleotide composition includes any one ormore of: base sequence (including length); pattern of chemicalmodifications to sugar and base moieties; pattern of backbone linkages;pattern of natural phosphate linkages, phosphorothioate linkages,phosphorothioate triester linkages, and combinations thereof, pattern ofbackbone chiral centers; pattern of stereochemistry (Rp/Sp) of chiralinternucleotidic linkages; pattern of backbone phosphorus modifications;pattern of modifications on the internucleotidic phosphorus atom, suchas —S⁻, and -L-R¹ of formula I.

Among other things, the present disclosure provides new compositionsthat are or contain particular stereoisomers of oligonucleotides ofinterest. In some embodiments, a particular stereoisomer may be defined,for example, by its base sequence, its length, its pattern of backbonelinkages, and its pattern of backbone chiral centers. As is understoodin the art, in some embodiments, base sequence may refer to the identityand/or modification status of nucleoside residues (e.g., of sugar and/orbase components, relative to standard naturally occurring nucleotidessuch as adenine, cytosine, guanosine, thymine, and uracil) in anoligonucleotide and/or to the hybridization character (i.e., the abilityto hybridize with particular complementary residues) of such residues.In some embodiments, oligonucleotides in provided compositions comprisesugar modifications, e.g., 2′-modifications, at e.g., a wing region. Insome embodiments, oligonucleotides in provided compositions comprise aregion in the middle, e.g., a core region, that has no sugarmodifications.

The present disclosure demonstrates, among other things, that individualstereoisomers of a particular oligonucleotide can show differentstability and/or activity (e.g., functional and/or toxicity properties)from each other. Moreover, the present disclosure demonstrates thatstability and/or activity improvements achieved through inclusion and/orlocation of particular chiral structures within an oligonucleotide canbe comparable to, or even better than those achieved through use ofparticular backbone linkages, residue modifications, etc. (e.g., throughuse of certain types of modified phosphates [e.g., phosphorothioate,substituted phosphorothioate, etc.], sugar modifications [e.g.,2′-modifications, etc.], and/or base modifications [e.g., methylation,etc.]).

Among other things, the present disclosure recognizes that, in someembodiments, properties (e.g., stability and/or activities) of anoligonucleotide can be adjusted by optimizing its pattern of backbonechiral centers, optionally in combination with adjustment/optimizationof one or more other features (e.g., linkage pattern, nucleosidemodification pattern, etc.) of the oligonucleotide. In some embodiments,the present disclosure provides oligonucleotide compositions wherein theoligonucleotides comprise nucleoside modifications, chiralinternucleotidic linkages and natural phosphate linkages. For example,WV-1092 comprises 2′-OMe modifications, phosphate linkages in its 5′-and 3′-wing regions, and phosphorothioate linkages in its core regions.

In some embodiments, the present disclosure demonstrates that stabilityimprovements achieved through inclusion and/or location of particularchiral structures within an oligonucleotide can be comparable to, oreven better than those achieved through use of modified backbonelinkages, bases, and/or sugars (e.g., through use of certain types ofmodified phosphates, 2′-modifications, base modifications, etc.). Thepresent disclosure, in some embodiments, also demonstrates that activityimprovements achieved through inclusion and/or location of particularchiral structures within an oligonucleotide can be comparable to, oreven better than those achieved through use of modified backbonelinkages, bases, and/or sugars (e.g., through use of certain types ofmodified phosphates, 2′-modifications, base modifications, etc.).

In some embodiments, inclusion and/or location of particular chirallinkages within an oligonucleotide can surprisingly change the cleavagepattern of a nucleic acid polymer when such an oligonucleotide isutilized for cleaving said nucleic acid polymer. For example, in someembodiments, a pattern of backbone chiral centers provides unexpectedlyhigh cleavage efficiency of a target nucleic acid polymer. In someembodiments, a pattern of backbone chiral centers provides new cleavagesites. In some embodiments, a pattern of backbone chiral centersprovides fewer cleavage sites, for example, by blocking certain existingcleavage sites. Even more unexpectedly, in some embodiments, a patternof backbone chiral centers provides cleavage at only one site of atarget nucleic acid polymer within the sequence that is complementary toan oligonucleotide utilized for cleavage. In some embodiments, highercleavage efficiency is achieved by selecting a pattern of backbonechiral centers to minimize the number of cleavage sites.

In some embodiments, the present disclosure provides compositions ofoligonucleotides, wherein the oligonucleotides have a common pattern ofbackbone chiral centers which, unexpectedly, greatly enhances thestability and/or biological activity of the oligonucleotides. In someembodiments, a pattern of backbone chiral centers provides increasedstability. In some embodiments, a pattern of backbone chiral centersprovides surprisingly increased activity. In some embodiments, a patternof backbone chiral centers provides increased stability and activity. Insome embodiments, when an oligonucleotide is utilized to cleave anucleic acid polymer, a pattern of backbone chiral centers, surprisinglyby itself, changes the cleavage pattern of a target nucleic acidpolymer. In some embodiments, a pattern of backbone chiral centerseffectively prevents cleavage at secondary sites. In some embodiments, apattern of backbone chiral centers creates new cleavage sites. In someembodiments, a pattern of backbone chiral centers minimizes the numberof cleavage sites. In some embodiments, a pattern of backbone chiralcenters minimizes the number of cleavage sites so that a target nucleicacid polymer is cleaved at only one site within the sequence of thetarget nucleic acid polymer that is complementary to the oligonucleotide(e.g., cleavage at other sites cannot be readily detected by a certainmethod; in some embodiments, greater than 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% cleavage occurs at such a site). In someembodiments, a pattern of backbone chiral centers enhances cleavageefficiency at a cleavage site. In some embodiments, a pattern ofbackbone chiral centers of the oligonucleotide improves cleavage of atarget nucleic acid polymer. In some embodiments, a pattern of backbonechiral centers increases selectivity. In some embodiments, a pattern ofbackbone chiral centers minimizes off-target effect. In someembodiments, a pattern of backbone chiral centers increase selectivity,e.g., cleavage selectivity between two target sequences differing onlyby a single nucleotide polymorphism (SNP). In some embodiments, apattern of backbone chiral centers increase cleavage at a cleavage siteof a stereorandom or DNA oligonucleotide composition. In someembodiments, a pattern of backbone chiral centers increase cleavage at amajor cleavage site of a stereorandom or DNA oligonucleotidecomposition. In some embodiments, such a site is a major cleavage siteof oligonucleotides having the pattern of backbone chiral centers. Insome embodiments, a site is considered a major site if it is a sitehaving the most, or the second, third, fourth or fifth most cleavage, ora site where greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of cleavage occurs. In some embodiments, a pattern of backbone chiralcenters comprises or is (Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m). In someembodiments, a pattern of backbone chiral centers comprises or is(Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m),wherein m>2. In some embodiments, a pattern of backbone chiral centerscomprises or is (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein n is 1, t>1, and m>2. In someembodiments, m>3. In some embodiments, m>4.

In some embodiments, the present disclosure recognizes that chemicalmodifications, such as modifications of nucleosides and internucleotidiclinkages, can provide enhanced properties. In some embodiments, thepresent disclosure demonstrates that combinations of chemicalmodifications and stereochemistry can provide unexpected, greatlyimproved properties (e.g., bioactivity, selectivity, etc.). In someembodiments, chemical combinations, such as modifications of sugars,bases, and/or internucleotidic linkages, are combined withstereochemistry patterns, e.g., (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m), to provideoligonucleotides and compositions thereof with surprisingly enhancedproperties. In some embodiments, a provided oligonucleotide compositionis chirally controlled, and comprises a combination of 2′-modificationof one or more sugar moieties, one or more natural phosphate linkages,one or more phosphorothioate linkages, and a stereochemistry pattern of(Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m),wherein m>2. In some embodiments, n is 1, t>1, and m>2. In someembodiments, m>3. In some embodiments, m>4.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotidesdefined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, a common base sequence and length may be referredto as a common base sequence. In some embodiments, oligonucleotideshaving a common base sequence may have the same pattern of nucleosidemodifications, e.g., sugar modifications, base modifications, etc. Insome embodiments, a pattern of nucleoside modifications may berepresented by a combination of locations and modifications. Forexample, for WV-1092, the pattern of nucleoside modifications is5×2′-OMe (2′-OMe modification on sugar moieties)-DNA (no2′-modifications on the sugar moiety)-5×2′-OMe from the 5′-end to the3′-end. In some embodiments, a pattern of backbone linkages compriseslocations and types (e.g., phosphate, phosphorothioate, substitutedphosphorothioate, etc.) of each internucleotidic linkages. In someembodiments, an oligonucleotide can have a specified pattern of backbonelinkages. In some embodiments, an oligonucleotide has a pattern ofbackbone linkages of _(n)PS-_(n)PO-_(n)PS-_(n)PO-_(n)PS, wherein PO isphosphate (phosphorodiester), PS is phosphorothioate, and n is 1-15, andeach occurrence of n can be the same or different. In some embodiments,at least one n is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20. In some embodiments, at least one n forPS is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. In some embodiments, the n for the PS between the twoPO is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20. In some embodiments, n is greater than 5. In someembodiments, n is greater than 6. In some embodiments, n is greater than7. In some embodiments, n is greater than 8. In some embodiments, n isgreater than 9. In some embodiments, n is greater than 10. In someembodiments, n is greater than 11. In some embodiments, n is greaterthan 12. In some embodiments, n is greater than 13. In some embodiments,n is greater than 14. In some embodiments, n is greater than 15. In someembodiments, an oligonucleotide has a pattern of backbone linkages of1-5PS-1-7PO-5-15PS-1-7 PO-1-5PS (meaning 1 to 5 phosphorothioates, 1 to7 phosphates, 5 to 15 phosphorothioates, 1 to 7 phosphates, and 1 to 5phosphorothioates). In some embodiments, the oligonucleotide has apattern of backbone linkages, from 5′ to 3′, of 1PS-3PO-11PS-3PO-1PS(meaning 1 phosphorothioate, 3 phosphates, 11 phosphorothioates, 3phosphates, and 1 phosphorothioate, and which can alternatively berepresented as PS₁PO₃PS₁₁PO₃PS₁). For example, for WV-1092, the patternof backbone linkages is 1PS-3PO-11PS-3PO-1PS from the 5′-end to the3′-end. In some embodiments, an oligonucleotide has a pattern ofbackbone linkages of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS, wherein each PS isSp except for one Rp. In some embodiments, the oligonucleotide has apattern of backbone linkages of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS, whereineach PS is Sp except one PS at any position from the 5^(th) to 15^(th)PS is Rp. In some embodiments, the oligonucleotide has a pattern ofbackbone linkages of 1-5PS-1-7PO-5-15PS-1-7PO-1-5PS, wherein each PS isSp except that the 10^(th) PS counting from the 5′ end is Rp. In someembodiments, the oligonucleotide has a pattern of backbone linkages of1-5PS-1-7PO-5-15PS-1-7PO-1-5PS, wherein each PS is Sp except that the9^(th) counting from the 5′ end PS is Rp. In some embodiments, theoligonucleotide has a pattern of backbone linkages of1-5PS-1-7PO-5-15PS-1-7PO-1-5PS, wherein each PS is Sp except that the11^(th) PS counting from the 5′ end is Rp. A pattern of backbone chiralcenters of an oligonucleotide can be designated by a combination oflinkage phosphorus stereochemistry (Rp/Sp) from 5′ to 3′. For example,WV-1092 has a pattern of 1S-3PO (phosphate)-8S-1R-2S-3PO-1S, and WV-937has a pattern of 12S-1R-6S. In some embodiments, all non-chiral linkages(e.g., PO) may be omitted when describing a pattern of backbone chiralcenters. As exemplified above, locations of non-chiral linkages may beobtained, for example, from pattern of backbone linkages. Any sequencedisclosed herein can be combined with any patterns of backbone linkagesand/or any patterns of backbone chiral centers disclosed herein. Basesequences, patterns of backbone linkages, patterns of stereochemistry(e.g., Rp or Sp), patterns of base modifications, patterns of backbonechiral centers, etc. are presented in 5′ to 3′ direction unlessotherwise indicated.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

An example substantially racemic preparation of oligonucleotides is thepreparation of phosphorothioate oligonucleotides through sulfurizingphosphite triesters from commonly used phosphoramidite oligonucleotidesynthesis with either tetraethylthiuram disulfide or (TETD) or 3H-1,2-bensodithiol-3-one 1, 1-dioxide (BDTD), a well-known process in theart. In some embodiments, substantially racemic preparation ofoligonucleotides provides substantially racemic oligonucleotidecompositions (or chirally uncontrolled oligonucleotide compositions).

As understood by a person having ordinary skill in the art, astereorandom or racemic preparation of oligonucleotides is prepared bynon-stereoselective and/or low-stereoselective coupling of nucleotidemonomers, typically without using any chiral auxiliaries, chiralmodification reagents, and/or chiral catalysts. In some embodiments, ina substantially racemic (or chirally uncontrolled) preparation ofoligonucleotides, all or most coupling steps are not chirally controlledin that the coupling steps are not specifically conducted to provideenhanced stereoselectivity. An example substantially racemic preparationof oligonucleotides is the preparation of phosphorothioateoligonucleotides through sulfurizing phosphite triesters from commonlyused phosphoramidite oligonucleotide synthesis with eithertetraethylthiuram disulfide or (TETD) or 3H-1, 2-bensodithiol-3-one 1,1-dioxide (BDTD), a well-known process in the art. In some embodiments,substantially racemic preparation of oligonucleotides providessubstantially racemic oligonucleotide compositions (or chirallyuncontrolled oligonucleotide compositions). In some embodiments, atleast one coupling of a nucleotide monomer has a diastereoselectivitylower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3,98:2, or 99:1. In some embodiments, at least two couplings of anucleotide monomer have a diastereoselectivity lower than about 60:40,70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In someembodiments, at least three couplings of a nucleotide monomer have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least fourcouplings of a nucleotide monomer have a diastereoselectivity lower thanabout 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or99:1. In some embodiments, at least five couplings of a nucleotidemonomer have a diastereoselectivity lower than about 60:40, 70:30,80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In someembodiments, in a stereorandom or racemic preparations, at least oneinternucleotidic linkage has a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, at least two internucleotidic linkages have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least threeinternucleotidic linkages have a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, at least four internucleotidic linkages have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least fiveinternucleotidic linkages have a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, a diastereoselectivity is lower than about 60:40. Insome embodiments, a diastereoselectivity is lower than about 70:30. Insome embodiments, a diastereoselectivity is lower than about 80:20. Insome embodiments, a diastereoselectivity is lower than about 90:10. Insome embodiments, a diastereoselectivity is lower than about 91:9. Insome embodiments, a diastereoselectivity is lower than about 92:8. Insome embodiments, a diastereoselectivity is lower than about 93:7. Insome embodiments, a diastereoselectivity is lower than about 94:6. Insome embodiments, a diastereoselectivity is lower than about 95:5. Insome embodiments, a diastereoselectivity is lower than about 96:4. Insome embodiments, a diastereoselectivity is lower than about 97:3. Insome embodiments, a diastereoselectivity is lower than about 98:2. Insome embodiments, a diastereoselectivity is lower than about 99:1. Insome embodiments, at least one coupling has a diastereoselectivity lowerthan about 90:10. In some embodiments, at least two couplings have adiastereoselectivity lower than about 90:10. In some embodiments, atleast three couplings have a diastereoselectivity lower than about90:10. In some embodiments, at least four couplings have adiastereoselectivity lower than about 90:10. In some embodiments, atleast five couplings have a diastereoselectivity lower than about 90:10.In some embodiments, at least one internucleotidic linkage has adiastereoselectivity lower than about 90:10. In some embodiments, atleast two internucleotidic linkages have a diastereoselectivity lowerthan about 90:10. In some embodiments, at least three internucleotidiclinkages have a diastereoselectivity lower than about 90:10. In someembodiments, at least four internucleotidic linkages have adiastereoselectivity lower than about 90:10. In some embodiments, atleast five internucleotidic linkages have a diastereoselectivity lowerthan about 90:10.

As understood by a person having ordinary skill in the art, in someembodiments, diastereoselectivity of a coupling or a linkage can beassessed through the diastereoselectivity of a dimer formation under thesame or comparable conditions, wherein the dimer has the same 5′- and3′-nucleosides and internucleotidic linkage. For example,diastereoselectivity of the underlined coupling or linkage in WV-1092mG*SmGmCmAmC*SA*SA*SG*SG*S G*SC*SA*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO:14) can be assessed from coupling two G moieties under the same orcomparable conditions, e.g., monomers, chiral auxiliaries, solvents,activators, temperatures, etc.

In some embodiments, the present disclosure provides chirally controlled(and/or stereochemically pure) oligonucleotide compositions comprisingoligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that atleast about 10% of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide type.In some embodiments, the present disclosure provides chirally controlledoligonucleotide composition of oligonucleotides in that the compositionis enriched, relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide typethat share:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

In some embodiments, oligonucleotides having a common base sequence andlength, a common pattern of backbone linkages, and a common pattern ofbackbone chiral centers have a common pattern of backbone phosphorusmodifications and a common pattern of base modifications. In someembodiments, oligonucleotides having a common base sequence and length,a common pattern of backbone linkages, and a common pattern of backbonechiral centers have a common pattern of backbone phosphorusmodifications and a common pattern of nucleoside modifications. In someembodiments, oligonucleotides having a common base sequence and length,a common pattern of backbone linkages, and a common pattern of backbonechiral centers have identical structures.

In some embodiments, oligonucleotides of an oligonucleotide type have acommon pattern of backbone phosphorus modifications and a common patternof sugar modifications. In some embodiments, oligonucleotides of anoligonucleotide type have a common pattern of backbone phosphorusmodifications and a common pattern of base modifications. In someembodiments, oligonucleotides of an oligonucleotide type have a commonpattern of backbone phosphorus modifications and a common pattern ofnucleoside modifications. In some embodiments, oligonucleotides of anoligonucleotide type are identical.

In some embodiments, a chirally controlled oligonucleotide compositionis a substantially pure preparation of an oligonucleotide type in thatoligonucleotides in the composition that are not of the oligonucleotidetype are impurities form the preparation process of said oligonucleotidetype, in some case, after certain purification procedures.

In some embodiments, at least about 20% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 25% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 30% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 35% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 40% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 45% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 50% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 55% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 60% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 65% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 70% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 75% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 80% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 85% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 90% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 92% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 94% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 95% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, greater thanabout 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, purity of achirally controlled oligonucleotide composition of an oligonucleotidecan be expressed as the percentage of oligonucleotides in thecomposition that have a common base sequence and length, a commonpattern of backbone linkages, and a common pattern of backbone chiralcenters.

In some embodiments, oligonucleotides having a common base sequence andlength, a common pattern of backbone linkages, and a common pattern ofbackbone chiral centers have a common pattern of backbone phosphorusmodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of nucleosidemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of sugarmodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of basemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of nucleosidemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers are identical.

In some embodiments, oligonucleotides in provided compositions have acommon pattern of backbone phosphorus modifications. In someembodiments, a common base sequence is a base sequence of anoligonucleotide type. In some embodiments, a provided composition is anoligonucleotide composition that is chirally controlled in that thecomposition contains a predetermined level of oligonucleotides of anindividual oligonucleotide type, wherein an oligonucleotide type isdefined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

As noted above and understood in the art, in some embodiments, basesequence of an oligonucleotide may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in theoligonucleotide and/or to the hybridization character (i.e., the abilityto hybridize with particular complementary residues) of such residues.

In some embodiments, a particular oligonucleotide type may be defined by

1A) base identity;

1B) pattern of base modification;

1C) pattern of sugar modification;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

Thus, in some embodiments, oligonucleotides of a particular type mayshare identical bases but differ in their pattern of base modificationsand/or sugar modifications. In some embodiments, oligonucleotides of aparticular type may share identical bases and pattern of basemodifications (including, e.g., absence of base modification), butdiffer in pattern of sugar modifications.

In some embodiments, oligonucleotides of a particular type are identicalin that they have the same base sequence (including length), the samepattern of chemical modifications to sugar and base moieties, the samepattern of backbone linkages (e.g., pattern of natural phosphatelinkages, phosphorothioate linkages, phosphorothioate triester linkages,and combinations thereof), the same pattern of backbone chiral centers(e.g., pattern of stereochemistry (Rp/Sp) of chiral internucleotidiclinkages), and the same pattern of backbone phosphorus modifications(e.g., pattern of modifications on the internucleotidic phosphorus atom,such as —S⁻, and -L-R¹ of formula I).

In some embodiments, purity of a chirally controlled oligonucleotidecomposition of an oligonucleotide type is expressed as the percentage ofoligonucleotides in the composition that are of the oligonucleotidetype. In some embodiments, at least about 10% of the oligonucleotides ina chirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 20% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 30% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 40% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 50% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 60% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 70% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 80% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 90% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 92% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 94% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 95% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 96% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 97% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 98% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 99% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type.

In some embodiments, purity of a chirally controlled oligonucleotidecomposition can be controlled by stereoselectivity of each coupling stepin its preparation process. In some embodiments, a coupling step has astereoselectivity (e.g., diastereoselectivity) of 60% (60% of the newinternucleotidic linkage formed from the coupling step has the intendedstereochemistry). After such a coupling step, the new internucleotidiclinkage formed may be referred to have a 60% purity. In someembodiments, each coupling step has a stereoselectivity of at least 60%.In some embodiments, each coupling step has a stereoselectivity of atleast 70%. In some embodiments, each coupling step has astereoselectivity of at least 80%. In some embodiments, each couplingstep has a stereoselectivity of at least 85%. In some embodiments, eachcoupling step has a stereoselectivity of at least 90%. In someembodiments, each coupling step has a stereoselectivity of at least 91%.In some embodiments, each coupling step has a stereoselectivity of atleast 92%. In some embodiments, each coupling step has astereoselectivity of at least 93%. In some embodiments, each couplingstep has a stereoselectivity of at least 94%. In some embodiments, eachcoupling step has a stereoselectivity of at least 95%. In someembodiments, each coupling step has a stereoselectivity of at least 96%.In some embodiments, each coupling step has a stereoselectivity of atleast 97%. In some embodiments, each coupling step has astereoselectivity of at least 98%. In some embodiments, each couplingstep has a stereoselectivity of at least 99%. In some embodiments, eachcoupling step has a stereoselectivity of at least 99.5%. In someembodiments, each coupling step has a stereoselectivity of virtually100%. In some embodiments, a coupling step has a stereoselectivity ofvirtually 100% in that all detectable product from the coupling step byan analytical method (e.g., NMR, HPLC, etc) has the intendedstereoselectivity.

Among other things, the present disclosure recognizes that combinationsof oligonucleotide structural elements (e.g., patterns of chemicalmodifications, backbone linkages, backbone chiral centers, and/orbackbone phosphorus modifications) can provide surprisingly improvedproperties such as bioactivities.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a predetermined level of oligonucleotides whichcomprise one or more wing regions and a common core region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

In some embodiments, a wing region comprises a structural feature thatis not in a core region. In some embodiments, a wing and core can bedefined by any structural elements, e.g., base modifications (e.g.,methylated/non-methylated, methylation at position 1/methylation atposition 2, etc.), sugar modifications (e.g., modified/non-modified,2′-modification/another type of modification, one type of2′-modification/another type of 2′-modification, etc.), backbone linkagetypes (e.g., phosphate/phosphorothioate, phosphorothioate/substitutedphosphorothioate, etc.), backbone chiral center stereochemistry (e.g.,all Sp/all Rp, (SpRp) repeats/all Rp, etc.), backbone phosphorusmodification types (e.g., s1/s2, s1/s3, etc.), etc.

In some embodiments, a wing and core is defined by nucleosidemodifications, wherein a wing comprises a nucleoside modification thatthe core region does not have. In some embodiments, a wing and core isdefined by sugar modifications, wherein a wing comprises a sugarmodification that the core region does not have. In some embodiments, asugar modification is a 2′-modification. In some embodiments, a sugarmodification is 2′-OR¹. In some embodiments, a sugar modification is2′-MOE. In some embodiments, a sugar modification is 2′-OMe.Additionally example sugar modifications are described in the presentdisclosure.

In some embodiments, oligonucleotides in provided compositions have awing-core structure (hemimer). In some embodiments, oligonucleotides inprovided compositions have a wing-core structure of nucleosidemodifications. In some embodiments, oligonucleotides in providedcompositions have a core-wing structure (another type of hemimer). Insome embodiments, oligonucleotides in provided compositions have acore-wing structure of nucleoside modifications. In some embodiments,oligonucleotides in provided compositions have a wing-core-wingstructure (gapmer). In some embodiments, oligonucleotides in providedcompositions have a wing-core-wing structure of nucleosidemodifications. In some embodiments, a wing and core is defined bymodifications of the sugar moieties. In some embodiments, a wing andcore is defined by modifications of the base moieties. In someembodiments, each sugar moiety in the wing region has the same2′-modification which is not found in the core region. In someembodiments, each sugar moiety in the wing region has the same2′-modification which is different than any sugar modifications in thecore region. In some embodiments, a core region has no sugarmodification. In some embodiments, each sugar moiety in the wing regionhas the same 2′-modification, and the core region has no2′-modifications. In some embodiments, when two or more wings arepresent, each wing is defined by its own modifications. In someembodiments, each wing has its own characteristic sugar modification. Insome embodiments, each wing has the same characteristic sugarmodification differentiating it from a core. In some embodiments, eachwing sugar moiety has the same modification. In some embodiments, eachwing sugar moiety has the same 2′-modification. In some embodiments,each sugar moiety in a wing region has the same 2′-modification, yet thecommon 2′-modification in a first wing region can either be the same asor different from the common 2′-modification in a second wing region. Insome embodiments, each sugar moiety in a wing region has the same2′-modification, and the common 2′-modification in a first wing regionis the same as the common 2′-modification in a second wing region. Insome embodiments, each sugar moiety in a wing region has the same2′-modification, and the common 2′-modification in a first wing regionis different from the common 2′-modification in a second wing region.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are antisense oligonucleotides(e.g., chiromersen). In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are siRNA oligonucleotides.In some embodiments, a provided chirally controlled oligonucleotidecomposition is of oligonucleotides that can be antisenseoligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir,ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide,triplex forming oligonucleotide, aptamer or adjuvant. In someembodiments, a chirally controlled oligonucleotide composition is ofantisense oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of antagomir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofmicroRNA oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of pre-microRNA oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofantimir oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of supermir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofribozyme oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of Ul adaptor oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is of RNAactivator oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of RNAi agent oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofdecoy oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of triplex forming oligonucleotides. Insome embodiments, a chirally controlled oligonucleotide composition isof aptamer oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of adjuvant oligonucleotides.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that includeone or more modified backbone linkages, bases, and/or sugars.

In some embodiments, a provided oligonucleotide comprises one or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide comprises two or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide comprisesthree or more chiral, modified phosphate linkages. In some embodiments,a provided oligonucleotide comprises four or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotidecomprises five or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25chiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 5 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises6 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 7 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 8 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 9 or more chiral,modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 10 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises11 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 12 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 13 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 14 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 15 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises16 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 17 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 18 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 19 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 20 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises21 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 22 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 23 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 24 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 25 or more chiral, modified phosphatelinkages.

In some embodiments, a provided oligonucleotide comprises at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% chiral, modified phosphate linkages. Examplesuch chiral, modified phosphate linkages are described above and herein.In some embodiments, a provided oligonucleotide comprises at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% chiral, modified phosphate linkages in theSp configuration.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 80%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 85%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 90%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 91%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 92%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 93%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 94%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 95%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 96%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 97%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 98%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 99%.

In some embodiments, a chiral, modified phosphate linkage is a chiralphosphorothioate linkage, i.e., phosphorothioate internucleotidiclinkage. In some embodiments, a provided oligonucleotide comprises atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioateinternucleotidic linkages. In some embodiments, all chiral, modifiedphosphate linkages are chiral phosphorothioate internucleotidiclinkages. In some embodiments, at least about 10, 20, 30, 40, 50, 60,70, 80, or 90% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 10% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 20% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 40%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 50% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 60% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 70% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 95% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 10% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 20% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 30% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 50%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 60% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 70% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 80% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 95%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 10%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 20% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 30% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 40% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 50%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 60% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 70% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 80% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 95% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, a provided oligonucleotide has only one Rp chiralphosphorothioate internucleotidic linkages. In some embodiments, aprovided oligonucleotide has only one Rp chiral phosphorothioateinternucleotidic linkages, wherein all internucleotide linkages arechiral phosphorothioate internucleotidic linkages. In some embodiments,a chiral phosphorothioate internucleotidic linkage is a chiralphosphorothioate diester linkage. In some embodiments, each chiralphosphorothioate internucleotidic linkage is independently a chiralphosphorothioate diester linkage. In some embodiments, eachinternucleotidic linkage is independently a chiral phosphorothioatediester linkage. In some embodiments, each internucleotidic linkage isindependently a chiral phosphorothioate diester linkage, and only one isRp.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that containone or more modified bases. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are ofoligonucleotides that contain no modified bases. Example such modifiedbases are described above and herein.

In some embodiments, oligonucleotides of provided compositions compriseat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. Insome embodiments, oligonucleotides of provided compositions comprise atleast one natural phosphate linkage. In some embodiments,oligonucleotides of provided compositions comprise at least two naturalphosphate linkages. In some embodiments, oligonucleotides of providedcompositions comprise at least three natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise at leastfour natural phosphate linkages. In some embodiments, oligonucleotidesof provided compositions comprise at least five natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least six natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sevennatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise at least eight natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least nine natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least ten naturalphosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise onenatural phosphate linkage. In some embodiments, oligonucleotides ofprovided compositions comprise two natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise threenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise four natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise fivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise six natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise sevennatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise eight natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise ninenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise ten natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions compriseat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least two consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise at leastthree consecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least fourconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least fiveconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sixconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sevenconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least eightconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least nineconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least tenconsecutive natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphate linkages. Insome embodiments, oligonucleotides of provided compositions comprise twoconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise three consecutivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise four consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise five consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise sixconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise seven consecutivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise eight consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise nine consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise tenconsecutive natural phosphate linkages.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 8 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 9 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 10 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 11 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 12bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 13 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 14 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 15 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 16 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 17bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 18 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 19 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 20 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 21 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 22bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 23 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 24 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 25 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 30, 35, 40, 45, 50, 55, 60,65, 70, or 75 bases.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations comprise oligonucleotides containingone or more residues which are modified at the sugar moiety. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations comprise oligonucleotides containing one or more residueswhich are modified at the 2′ position of the sugar moiety (referred toherein as a “2′-modification”). Examples of such modifications aredescribed above and herein and include, but are not limited to, 2′-OMe,2′-MOE, 2′-LNA, 2′-F, FRNA, FANA, S-cEt, etc. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationscomprise oligonucleotides containing one or more residues which are2′-modified. For example, in some embodiments, provided oligonucleotidescontain one or more residues which are 2′-O-methoxyethyl(2′-MOE)-modified residues. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations compriseoligonucleotides which do not contain any 2′-modifications. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are oligonucleotides which do not contain any 2′-MOEresidues. That is, in some embodiments, provided oligonucleotides arenot MOE-modified. Additional example sugar modifications are describedin the present disclosure.

In some embodiments, provided oligonucleotides are of a general motif ofwing-core or core-wing (hemimer, also represented herein generally asX—Y or Y—X, respectively). In some embodiments, providedoligonucleotides are of a general motif of wing-core-wing (gapmer, alsorepresented herein generically as X—Y—X). In some embodiments, each wingindependently contains one or more residues having a particularmodification, which modification is absent from the core “Y” portion. Insome embodiments, each wing independently contains one or more residueshaving a particular nucleoside modification, which modification isabsent from the core “Y” portion. In some embodiments, each wingindependently contains one or more residues having a particular basemodification, which modification is absent from the core “Y” portion. Insome embodiments, each wing independently contains one or more residueshaving a particular sugar modification, which modification is absentfrom the core “Y” portion. Example sugar modifications are widely knownin the art. In some embodiments, a sugar modification is a modificationselected from those modifications described in U.S. Pat. No. 9,006,198,which sugar modifications are incorporated herein by references.Additional example sugar modifications are described in the presentdisclosure. In some embodiment, each wing contains one or more residueshaving a 2′ modification that is not present in the core portion. Insome embodiments, a 2′-modification is 2′-OR¹, wherein R¹ is as definedand described in the present disclosure.

In some embodiments, provided oligonucleotides have a wing-core motifrepresented as X—Y, or a core-wing motif represented as Y—X, wherein theresidues at the “X” portion are sugar modified residues of a particulartype and the residues in the core “Y” portion are not sugar modifiedresidues of the same particular type. In some embodiments, providedoligonucleotides have a wing-core-wing motif represented as X—Y—X,wherein the residues at each “X” portion are sugar modified residues ofa particular type and the residues in the core “Y” portion are not sugarmodified residues of the same particular type. In some embodiments,provided oligonucleotides have a wing-core motif represented as X—Y, ora core-wing motif represented as Y—X, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have awing-core motif represented as X—Y, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have acore-wing motif represented as Y—X, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have awing-core-wing motif represented as X—Y—X, wherein the residues at each“X” portion are 2′-modified residues of a particular type and theresidues in the core “Y” portion are not 2′-modified residues of thesame particular type. In some embodiments, provided oligonucleotideshave a wing-core motif represented as X—Y, wherein the residues at the“X” portion are 2′-modified residues of a particular type and theresidues in the core “Y” portion are 2′-deoxyribonucleoside. In someembodiments, provided oligonucleotides have a core-wing motifrepresented as Y—X, wherein the residues at the “X” portion are2′-modified residues of a particular type and the residues in the core“Y” portion are 2′-deoxyribonucleoside. In some embodiments, providedoligonucleotides have a wing-core-wing motif represented as X—Y—X,wherein the residues at each “X” portion are 2′-modified residues of aparticular type and the residues in the core “Y” portion are2′-deoxyribonucleoside. In some embodiments, provided oligonucleotideshave a wing-core-wing motif represented as X—Y—X, wherein the residuesat each “X” portion are 2′-modified residues of a particular type andthe residues in the core “Y” portion are 2′-deoxyribonucleoside. Forinstance, in some embodiments, provided oligonucleotides have awing-core-wing motif represented as X—Y—X, wherein the residues at each“X” portion are 2′-MOE-modified residues and the residues in the core“Y” portion are not 2′-MOE-modified residues. In some embodiments,provided oligonucleotides have a wing-core-wing motif represented asX—Y—X, wherein the residues at each “X” portion are 2′-MOE-modifiedresidues and the residues in the core “Y” portion are2′-deoxyribonucleoside. One of skill in the relevant arts will recognizethat all such 2′-modifications described above and herein arecontemplated in the context of such X—Y, Y—X and/or X—Y—X motifs.

In some embodiments, a wing has a length of one or more bases. In someembodiments, a wing has a length of two or more bases. In someembodiments, a wing has a length of three or more bases. In someembodiments, a wing has a length of four or more bases. In someembodiments, a wing has a length of five or more bases. In someembodiments, a wing has a length of six or more bases. In someembodiments, a wing has a length of seven or more bases. In someembodiments, a wing has a length of eight or more bases. In someembodiments, a wing has a length of nine or more bases. In someembodiments, a wing has a length of ten or more bases. In someembodiments, a wing has a length of 11 or more bases. In someembodiments, a wing has a length of 12 or more bases. In someembodiments, a wing has a length of 13 or more bases. In someembodiments, a wing has a length of 14 or more bases. In someembodiments, a wing has a length of 15 or more bases. In someembodiments, a wing has a length of 16 or more bases. In someembodiments, a wing has a length of 17 or more bases. In someembodiments, a wing has a length of 18 or more bases. In someembodiments, a wing has a length of 19 or more bases. In someembodiments, a wing has a length often or more bases.

In some embodiments, a wing has a length of one base. In someembodiments, a wing has a length of two bases. In some embodiments, awing has a length of three bases. In some embodiments, a wing has alength of four bases. In some embodiments, a wing has a length of fivebases. In some embodiments, a wing has a length of six bases. In someembodiments, a wing has a length of seven bases. In some embodiments, awing has a length of eight bases. In some embodiments, a wing has alength of nine bases. In some embodiments, a wing has a length of tenbases. In some embodiments, a wing has a length of 11 bases. In someembodiments, a wing has a length of 12 bases. In some embodiments, awing has a length of 13 bases. In some embodiments, a wing has a lengthof 14 bases. In some embodiments, a wing has a length of 15 bases. Insome embodiments, a wing has a length of 16 bases. In some embodiments,a wing has a length of 17 bases. In some embodiments, a wing has alength of 18 bases. In some embodiments, a wing has a length of 19bases. In some embodiments, a wing has a length of ten bases.

In some embodiments, a wing comprises one or more chiralinternucleotidic linkages. In some embodiments, a wing comprises one ormore natural phosphate linkages. In some embodiments, a wing comprisesone or more chiral internucleotidic linkages and one or more naturalphosphate linkages. In some embodiments, a wing comprises one or morechiral internucleotidic linkages and two or more natural phosphatelinkages. In some embodiments, a wing comprises one or more chiralinternucleotidic linkages and two or more natural phosphate linkages,wherein two or more natural phosphate linkages are consecutive. In someembodiments, a wing comprises no chiral internucleotidic linkages. Insome embodiments, each wing linkage is a natural phosphate linkage. Insome embodiments, a wing comprises no phosphate linkages. In someembodiments, each wing is independently a chiral internucleotidiclinkage.

In some embodiments, each wing independently comprises one or morechiral internucleotidic linkages. In some embodiments, each wingindependently comprises one or more natural phosphate linkages. In someembodiments, each wing independently comprises one or more chiralinternucleotidic linkages and one or more natural phosphate linkages. Insome embodiments, each wing independently comprises one or more chiralinternucleotidic linkages and two or more natural phosphate linkages. Insome embodiments, each wing independently comprises one or more chiralinternucleotidic linkages and two or more natural phosphate linkages,wherein two or more natural phosphate linkages are consecutive.

In some embodiments, each wing independently comprises at least onechiral internucleotidic linkage. In some embodiments, each wingindependently comprises at least two chiral internucleotidic linkages.In some embodiments, each wing independently comprises at least threechiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least four chiral internucleotidic linkages.In some embodiments, each wing independently comprises at least fivechiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least six chiral internucleotidic linkages.In some embodiments, each wing independently comprises at least sevenchiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least eight chiral internucleotidic linkages.In some embodiments, each wing independently comprises at least ninechiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least ten chiral internucleotidic linkages.In some embodiments, each wing independently comprises at least 11chiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least 12 chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least 13 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 14 chiral internucleotidic linkages. In someembodiments, each wing independently comprises at least 15 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 16 chiral internucleotidic linkages. In someembodiments, each wing independently comprises at least 17 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 18 chiral internucleotidic linkages. In someembodiments, each wing independently comprises at least 19 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 20 chiral internucleotidic linkages.

In some embodiments, each wing independently comprises one chiralinternucleotidic linkage. In some embodiments, each wing independentlycomprises two chiral internucleotidic linkages. In some embodiments,each wing independently comprises three chiral internucleotidiclinkages. In some embodiments, each wing independently comprises fourchiral internucleotidic linkages. In some embodiments, each wingindependently comprises five chiral internucleotidic linkages. In someembodiments, each wing independently comprises six chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises seven chiral internucleotidic linkages. In some embodiments,each wing independently comprises eight chiral internucleotidiclinkages. In some embodiments, each wing independently comprises ninechiral internucleotidic linkages. In some embodiments, each wingindependently comprises ten chiral internucleotidic linkages. In someembodiments, each wing independently comprises 11 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 12 chiral internucleotidic linkages. In some embodiments, eachwing independently comprises 13 chiral internucleotidic linkages. Insome embodiments, each wing independently comprises 14 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 15 chiral internucleotidic linkages. In some embodiments, eachwing independently comprises 16 chiral internucleotidic linkages. Insome embodiments, each wing independently comprises 17 chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 18 chiral internucleotidic linkages. In some embodiments, eachwing independently comprises 19 chiral internucleotidic linkages. Insome embodiments, each wing independently comprises 20 chiralinternucleotidic linkages.

In some embodiments, each wing independently comprises at least oneconsecutive natural phosphate linkage. In some embodiments, each wingindependently comprises at least two consecutive chiral internucleotidiclinkages. In some embodiments, each wing independently comprises atleast three consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises at least four consecutivechiral internucleotidic linkages. In some embodiments, each wingindependently comprises at least five consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least six consecutive chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least sevenconsecutive chiral internucleotidic linkages. In some embodiments, eachwing independently comprises at least eight consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least nine consecutive chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least tenconsecutive chiral internucleotidic linkages. In some embodiments, eachwing independently comprises at least 11 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 12 consecutive chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least 13consecutive chiral internucleotidic linkages. In some embodiments, eachwing independently comprises at least 14 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 15 consecutive chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least 16consecutive chiral internucleotidic linkages. In some embodiments, eachwing independently comprises at least 17 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises at least 18 consecutive chiral internucleotidic linkages. Insome embodiments, each wing independently comprises at least 19consecutive chiral internucleotidic linkages. In some embodiments, eachwing independently comprises at least 20 consecutive chiralinternucleotidic linkages.

In some embodiments, each wing independently comprises one consecutivenatural phosphate linkage. In some embodiments, each wing independentlycomprises two consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises three consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises four consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises five consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises six consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises seven consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises eight consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises nine consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises ten consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises 11 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 12 consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises 13 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 14 consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises 15 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 16 consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises 17 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 18 consecutive chiral internucleotidic linkages. In someembodiments, each wing independently comprises 19 consecutive chiralinternucleotidic linkages. In some embodiments, each wing independentlycomprises 20 consecutive chiral internucleotidic linkages.

In some embodiments, each wing independently comprises at least onenatural phosphate linkage. In some embodiments, each wing independentlycomprises at least two natural phosphate linkages. In some embodiments,each wing independently comprises at least three natural phosphatelinkages. In some embodiments, each wing independently comprises atleast four natural phosphate linkages. In some embodiments, each wingindependently comprises at least five natural phosphate linkages. Insome embodiments, each wing independently comprises at least six naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least seven natural phosphate linkages. In someembodiments, each wing independently comprises at least eight naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least nine natural phosphate linkages. In some embodiments,each wing independently comprises at least ten natural phosphatelinkages. In some embodiments, each wing independently comprises atleast 11 natural phosphate linkages. In some embodiments, each wingindependently comprises at least 12 natural phosphate linkages. In someembodiments, each wing independently comprises at least 13 naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least 14 natural phosphate linkages. In some embodiments,each wing independently comprises at least 15 natural phosphatelinkages. In some embodiments, each wing independently comprises atleast 16 natural phosphate linkages. In some embodiments, each wingindependently comprises at least 17 natural phosphate linkages. In someembodiments, each wing independently comprises at least 18 naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least 19 natural phosphate linkages. In some embodiments,each wing independently comprises at least 20 natural phosphatelinkages.

In some embodiments, each wing independently comprises one naturalphosphate linkage. In some embodiments, each wing independentlycomprises two natural phosphate linkages. In some embodiments, each wingindependently comprises three natural phosphate linkages. In someembodiments, each wing independently comprises four natural phosphatelinkages. In some embodiments, each wing independently comprises fivenatural phosphate linkages. In some embodiments, each wing independentlycomprises six natural phosphate linkages. In some embodiments, each wingindependently comprises seven natural phosphate linkages. In someembodiments, each wing independently comprises eight natural phosphatelinkages. In some embodiments, each wing independently comprises ninenatural phosphate linkages. In some embodiments, each wing independentlycomprises ten natural phosphate linkages. In some embodiments, each wingindependently comprises 11 natural phosphate linkages. In someembodiments, each wing independently comprises 12 natural phosphatelinkages. In some embodiments, each wing independently comprises 13natural phosphate linkages. In some embodiments, each wing independentlycomprises 14 natural phosphate linkages. In some embodiments, each wingindependently comprises 15 natural phosphate linkages. In someembodiments, each wing independently comprises 16 natural phosphatelinkages. In some embodiments, each wing independently comprises 17natural phosphate linkages. In some embodiments, each wing independentlycomprises 18 natural phosphate linkages. In some embodiments, each wingindependently comprises 19 natural phosphate linkages. In someembodiments, each wing independently comprises 20 natural phosphatelinkages.

In some embodiments, each wing independently comprises at least oneconsecutive natural phosphate linkage. In some embodiments, each wingindependently comprises at least two consecutive natural phosphatelinkages. In some embodiments, each wing independently comprises atleast three consecutive natural phosphate linkages. In some embodiments,each wing independently comprises at least four consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least five consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least six consecutivenatural phosphate linkages. In some embodiments, each wing independentlycomprises at least seven consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least eightconsecutive natural phosphate linkages. In some embodiments, each wingindependently comprises at least nine consecutive natural phosphatelinkages. In some embodiments, each wing independently comprises atleast ten consecutive natural phosphate linkages. In some embodiments,each wing independently comprises at least 11 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises at least 12 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least 13 consecutivenatural phosphate linkages. In some embodiments, each wing independentlycomprises at least 14 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least 15 consecutivenatural phosphate linkages. In some embodiments, each wing independentlycomprises at least 16 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least 17 consecutivenatural phosphate linkages. In some embodiments, each wing independentlycomprises at least 18 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises at least 19 consecutivenatural phosphate linkages. In some embodiments, each wing independentlycomprises at least 20 consecutive natural phosphate linkages.

In some embodiments, each wing independently comprises one consecutivenatural phosphate linkage. In some embodiments, each wing independentlycomprises two consecutive natural phosphate linkages. In someembodiments, each wing independently comprises three consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises four consecutive natural phosphate linkages. In someembodiments, each wing independently comprises five consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises six consecutive natural phosphate linkages. In someembodiments, each wing independently comprises seven consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises eight consecutive natural phosphate linkages. In someembodiments, each wing independently comprises nine consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises ten consecutive natural phosphate linkages. In someembodiments, each wing independently comprises 11 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises 12 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises 13 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises 14 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises 15 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises 16 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises 17 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises 18 consecutive natural phosphate linkages. In someembodiments, each wing independently comprises 19 consecutive naturalphosphate linkages. In some embodiments, each wing independentlycomprises 20 consecutive natural phosphate linkages.

In some embodiments, a wing comprises only one chiral internucleotidiclinkage. In some embodiments, a 5′-end wing comprises only one chiralinternucleotidic linkage. In some embodiments, a 5′-end wing comprisesonly one chiral internucleotidic linkage at the 5′-end of the wing. Insome embodiments, a 5′-end wing comprises only one chiralinternucleotidic linkage at the 5′-end of the wing, and the chiralinternucleotidic linkage is Rp. In some embodiments, a 5′-end wingcomprises only one chiral internucleotidic linkage at the 5′-end of thewing, and the chiral internucleotidic linkage is Sp. In someembodiments, a 3′-end wing comprises only one chiral internucleotidiclinkage at the 3′-end of the wing. In some embodiments, a 3′-end wingcomprises only one chiral internucleotidic linkage at the 3′-end of thewing, and the chiral internucleotidic linkage is Rp. In someembodiments, a 3′-end wing comprises only one chiral internucleotidiclinkage at the 3′-end of the wing, and the chiral internucleotidiclinkage is Sp.

In some embodiments, a wing comprises two or more natural phosphatelinkages. In some embodiments, all phosphate linkages within a wing areconsecutive, and there are no non-phosphate linkages between any twophosphate linkages within a wing.

In some embodiments, a linkage connecting a wing and a core isconsidered part of the core when describing linkages, e.g., linkagechemistry, linkage stereochemistry, etc. For example, in WV-1092, mG*SmGmCmAmC*SA* SA*SG*SG*SG*SC S A*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO: 15),the underlined linkages may be considered as part of the core (bold),its 5′-wing (having 2′-OMe on sugar moieties) has one single Spphosphorothioate linkages at its 5′-end, its 3′-wing (having 2′-OMe onsugar moieties) has one Sp phosphorothioate linkage at its 3′-end, andits core has no 2′-modifications on sugar).

In some embodiments, a 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, a 5′-internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, a 5′-internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, a 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, a 5′-internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage. In some embodiments, each 5′-internucleotidic linkage connectedto a sugar moiety without a 2′-modification is a modified linkage. Insome embodiments, each 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a linkage having the structure offormula I. In some embodiments, each 5′-internucleotidic linkageconnected to a sugar moiety without a 2′-modification isphosphorothioate linkage. In some embodiments, each 5′-internucleotidiclinkage connected to a sugar moiety without a 2′-modification is asubstituted phosphorothioate linkage. In some embodiments, each5′-internucleotidic linkage connected to a sugar moiety without a2′-modification is a phosphorothioate triester linkage.

In some embodiments, a 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, a 3′-internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, a 3′-internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, a 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, a 3′-internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage. In some embodiments, each 3′-internucleotidic linkage connectedto a sugar moiety without a 2′-modification is a modified linkage. Insome embodiments, each 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a linkage having the structure offormula I. In some embodiments, each 3′-internucleotidic linkageconnected to a sugar moiety without a 2′-modification isphosphorothioate linkage. In some embodiments, each 3′-internucleotidiclinkage connected to a sugar moiety without a 2′-modification is asubstituted phosphorothioate linkage. In some embodiments, each3′-internucleotidic linkage connected to a sugar moiety without a2′-modification is a phosphorothioate triester linkage.

In some embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are modified linkages. In someembodiments, both internucleotidic linkages connected to a sugar moietywithout a 2′-modification are linkage having the structure of formula I.In some embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are phosphorothioate linkages. In someembodiments, both internucleotidic linkages connected to a sugar moietywithout a 2′-modification are substituted phosphorothioate linkages. Insome embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are phosphorothioate triester linkages.In some embodiments, each internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, each internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, each internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, each internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, each internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage.

In some embodiments, a sugar moiety without a 2′-modification is a sugarmoiety found in a natural DNA nucleoside.

In some embodiments, for a wing-core-wing structure, the 5′-end wingcomprises only one chiral internucleotidic linkage. In some embodiments,for a wing-core-wing structure, the 5′-end wing comprises only onechiral internucleotidic linkage at the 5′-end of the wing. In someembodiments, for a wing-core-wing structure, the 3′-end wing comprisesonly one chiral internucleotidic linkage. In some embodiments, for awing-core-wing structure, the 3′-end wing comprises only one chiralinternucleotidic linkage at the 3′-end of the wing. In some embodiments,for a wing-core-wing structure, each wing comprises only one chiralinternucleotidic linkage. In some embodiments, for a wing-core-wingstructure, each wing comprises only one chiral internucleotidic linkage,wherein the 5′-end wing comprises only one chiral internucleotidiclinkage at its 5′-end; and the 3′-end wing comprises only one chiralinternucleotidic linkage at its 3′-end. In some embodiments, the onlychiral internucleotidic linkage in the 5′-wing is Rp. In someembodiments, the only chiral internucleotidic linkage in the 5′-wing isSp. In some embodiments, the only chiral internucleotidic linkage in the3′-wing is Rp. In some embodiments, the only chiral internucleotidiclinkage in the 3′-wing is Sp. In some embodiments, the only chiralinternucleotidic linkage in both the 5′- and the 3′-wings are Sp. Insome embodiments, the only chiral internucleotidic linkage in both the5′- and the 3′-wings are Rp. In some embodiments, the only chiralinternucleotidic linkage in the 5′-wing is Sp, and the only chiralinternucleotidic linkage in the 3′-wing is Rp. In some embodiments, theonly chiral internucleotidic linkage in the 5′-wing is Rp, and the onlychiral internucleotidic linkage in the 3′-wing is Sp.

In some embodiments, a wing comprises two chiral internucleotidiclinkages. In some embodiments, a wing comprises only two chiralinternucleotidic linkages, and one or more natural phosphate linkages.In some embodiments, a wing comprises only two chiral internucleotidiclinkages, and two or more natural phosphate linkages. In someembodiments, a wing comprises only two chiral internucleotidic linkages,and two or more consecutive natural phosphate linkages. In someembodiments, a wing comprises only two chiral internucleotidic linkages,and two consecutive natural phosphate linkages. In some embodiments, awing comprises only two chiral internucleotidic linkages, and threeconsecutive natural phosphate linkages. In some embodiments, a 5′-wing(to a core) comprises only two chiral internucleotidic linkages, one atits 5′-end and the other at its 3′-end, with one or more naturalphosphate linkages in between. In some embodiments, a 5′-wing (to acore) comprises only two chiral internucleotidic linkages, one at its5′-end and the other at its 3′-end, with two or more natural phosphatelinkages in between. In some embodiments, a 3′-wing (to a core)comprises only two chiral internucleotidic linkages, one at its 3′-endand the other at its 3′-end, with one or more natural phosphate linkagesin between. In some embodiments, a 3′-wing (to a core) comprises onlytwo chiral internucleotidic linkages, one at its 3′-end and the other atits 3′-end, with two or more natural phosphate linkages in between.

In some embodiments, a 5′-wing comprises only two chiralinternucleotidic linkages, one at its 5′-end and the other at its3′-end, with one or more natural phosphate linkages in between, and the3′-wing comprise only one internucleotidic linkage at its 3′-end. Insome embodiments, a 5′-wing (to a core) comprises only two chiralinternucleotidic linkages, one at its 5′-end and the other at its3′-end, with two or more natural phosphate linkages in between, and the3′-wing comprise only one internucleotidic linkage at its 3′-end. Insome embodiments, each chiral internucleotidic linkage independently hasits own stereochemistry. In some embodiments, both chiralinternucleotidic linkages in the 5′-wing have the same stereochemistry.In some embodiments, both chiral internucleotidic linkages in the5′-wing have different stereochemistry. In some embodiments, both chiralinternucleotidic linkages in the 5′-wing are Rp. In some embodiments,both chiral internucleotidic linkages in the 5′-wing are Sp. In someembodiments, chiral internucleotidic linkages in the 5′- and 3′-wingshave the same stereochemistry. In some embodiments, chiralinternucleotidic linkages in the 5′- and 3′-wings are Rp. In someembodiments, chiral internucleotidic linkages in the 5′- and 3′-wingsare Sp. In some embodiments, chiral internucleotidic linkages in the 5′-and 3′-wings have different stereochemistry.

In some embodiments, a core region has a length of one or more bases. Insome embodiments, a core region has a length of two or more bases. Insome embodiments, a core region has a length of three or more bases. Insome embodiments, a core region has a length of four or more bases. Insome embodiments, a core region has a length of five or more bases. Insome embodiments, a core region has a length of six or more bases. Insome embodiments, a core region has a length of seven or more bases. Insome embodiments, a core region has a length of eight or more bases. Insome embodiments, a core region has a length of nine or more bases. Insome embodiments, a core region has a length of ten or more bases. Insome embodiments, a core region has a length of 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, or more bases. In certain embodiments, a core regionhas a length of 11 or more bases. In certain embodiments, a core regionhas a length of 12 or more bases. In certain embodiments, a core regionhas a length of 13 or more bases. In certain embodiments, a core regionhas a length of 14 or more bases. In certain embodiments, a core regionhas a length of 15 or more bases. In certain embodiments, a core regionhas a length of 16 or more bases. In certain embodiments, a core regionhas a length of 17 or more bases. In certain embodiments, a core regionhas a length of 18 or more bases. In certain embodiments, a core regionhas a length of 19 or more bases. In certain embodiments, a core regionhas a length of 20 or more bases. In certain embodiments, a core regionhas a length of more than 20 bases. In certain embodiments, a coreregion has a length of 2 bases. In certain embodiments, a core regionhas a length of 3 bases. In certain embodiments, a core region has alength of 4 bases. In certain embodiments, a core region has a length of5 bases. In certain embodiments, a core region has a length of 6 bases.In certain embodiments, a core region has a length of 7 bases. Incertain embodiments, a core region has a length of 8 bases. In certainembodiments, a core region has a length of 9 bases. In certainembodiments, a core region has a length of 10 bases. In certainembodiments, a core region has a length of 11 bases. In certainembodiments, a core region has a length of 12 bases. In certainembodiments, a core region has a length of 13 bases. In certainembodiments, a core region has a length of 14 bases. In certainembodiments, a core region has a length of 15 bases. In certainembodiments, a core region has a length of 16 bases. In certainembodiments, a core region has a length of 17 bases. In certainembodiments, a core region has a length of 18 bases. In certainembodiments, a core region has a length of 19 bases. In certainembodiments, a core region has a length of 20 bases.

In some embodiments, a core comprises one or more chiralinternucleotidic linkages. In some embodiments, a core comprises one ormore natural phosphate linkages. In some embodiments, a coreindependently comprises one or more chiral internucleotidic linkages andone or more natural phosphate linkages. In some embodiments, a corecomprises no phosphate linkages. In some embodiments, each core linkageis a chiral internucleotidic linkage.

In some embodiments, a core comprises at least one natural phosphatelinkage. In some embodiments, a core comprises at least two chiralinternucleotidic linkages. In some embodiments, a core comprises atleast three chiral internucleotidic linkages. In some embodiments, acore comprises at least four chiral internucleotidic linkages. In someembodiments, a core comprises at least five chiral internucleotidiclinkages. In some embodiments, a core comprises at least six chiralinternucleotidic linkages. In some embodiments, a core comprises atleast seven chiral internucleotidic linkages. In some embodiments, acore comprises at least eight chiral internucleotidic linkages. In someembodiments, a core comprises at least nine chiral internucleotidiclinkages. In some embodiments, a core comprises at least ten chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 11 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 12 chiral internucleotidic linkages. In someembodiments, a core comprises at least 13 chiral internucleotidiclinkages. In some embodiments, a core comprises at least 14 chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 15 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 16 chiral internucleotidic linkages. In someembodiments, a core comprises at least 17 chiral internucleotidiclinkages. In some embodiments, a core comprises at least 18 chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 19 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 20 chiral internucleotidic linkages.

In some embodiments, a core comprises one natural phosphate linkage. Insome embodiments, a core comprises two chiral internucleotidic linkages.In some embodiments, a core comprises three chiral internucleotidiclinkages. In some embodiments, a core comprises four chiralinternucleotidic linkages. In some embodiments, a core comprises fivechiral internucleotidic linkages. In some embodiments, a core comprisessix chiral internucleotidic linkages. In some embodiments, a corecomprises seven chiral internucleotidic linkages. In some embodiments, acore comprises eight chiral internucleotidic linkages. In someembodiments, a core comprises nine chiral internucleotidic linkages. Insome embodiments, a core comprises ten chiral internucleotidic linkages.In some embodiments, a core comprises 11 chiral internucleotidiclinkages. In some embodiments, a core comprises 12 chiralinternucleotidic linkages. In some embodiments, a core comprises 13chiral internucleotidic linkages. In some embodiments, a core comprises14 chiral internucleotidic linkages. In some embodiments, a corecomprises 15 chiral internucleotidic linkages. In some embodiments, acore comprises 16 chiral internucleotidic linkages. In some embodiments,a core comprises 17 chiral internucleotidic linkages. In someembodiments, a core comprises 18 chiral internucleotidic linkages. Insome embodiments, a core comprises 19 chiral internucleotidic linkages.In some embodiments, a core comprises 20 chiral internucleotidiclinkages.

In some embodiments, a core region has a pattern of backbone chiralcenters comprising (Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m), wherein each ofm, n, t and Np is independently as defined and described in the presentdisclosure. In some embodiments, a core region has a pattern of backbonechiral centers comprising (Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m),(Np)_(t)(Rp)_(n)(Sp)_(m), or (Sp)_(t)(Rp)_(n)(Sp)_(m). In someembodiments, a core region has a pattern of backbone chiral centerscomprising (Sp)_(m)(Rp)_(n). In some embodiments, a core region has apattern of backbone chiral centers comprising (Sp)_(m)(Rp)_(n), whereinm>2 and n is 1. In some embodiments, a core region has a pattern ofbackbone chiral centers comprising (Rp)_(n)(Sp)_(m). In someembodiments, a core region has a pattern of backbone chiral centerscomprising (Rp)_(n)(Sp)_(m), wherein m>2 and n is 1. In someembodiments, a core region has a pattern of backbone chiral centerscomprising (Np)_(t)(Rp)_(n)(Sp)_(m). In some embodiments, a core regionhas a pattern of backbone chiral centers comprising(Np)_(t)(Rp)_(n)(Sp)_(m), wherein m>2 and n is 1. In some embodiments, acore region has a pattern of backbone chiral centers comprising(Np)_(t)(Rp)_(n)(Sp)_(m), wherein t>2, m>2 and n is 1. In someembodiments, a core region has a pattern of backbone chiral centerscomprising (Sp)_(t)(Rp)_(n)(Sp)_(m). In some embodiments, a core regionhas a pattern of backbone chiral centers comprising(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein m>2 and n is 1. In some embodiments, acore region has a pattern of backbone chiral centers comprising(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein t>2, m>2 and n is 1. Among otherthings, the present disclosure demonstrates that, in some embodiments,such patterns can provide and/or enhance controlled cleavage, improvedcleavage rate, selectivity, etc., of a target sequence, e.g., an RNAsequence. Example patterns of backbone chiral centers are described inthe present disclosure.

In some embodiments, at least 60% of the chiral internucleotidiclinkages in the core region are Sp. In some embodiments, at least 65% ofthe chiral internucleotidic linkages in the core region are Sp. In someembodiments, at least 66% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 67% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 70% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 75% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 80% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 85% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 90% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 95% of the chiralinternucleotidic linkages in the core region are Sp.

In some embodiments, a wing-core-wing (i.e., X—Y—X) motif is representednumerically as, e.g., 5-10-4, meaning the wing to the 5′-end of the coreis 5 bases in length, the core region is 10 bases in length, and thewing region to the 3′-end of the core is 4-bases in length. In someembodiments, a wing-core-wing motif is any of, e.g. 2-16-2, 3-14-3,4-12-4, 5-10-5, 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4-9-3, 4-9-4, 4-9-5,4-10-5, 4-11-4, 4-11-5, 5- 7-5, 5-8-6, 8-7-5, 7-7-6, 5-9-3, 5-9-5,5-10-4, 5-10-5, 6-7-6, 6-8-5, and 6-9-2, etc. In certain embodiments, awing-core-wing motif is 5-10-5. In certain embodiments, a wing-core-wingmotif is 7-7-6. In certain embodiments, a wing-core-wing motif is 8-7-5.

In some embodiments, a wing-core motif is 5-15, 6-14, 7-13, 8-12, 9-12,etc. In some embodiments, a core-wing motif is 5-15, 6-14, 7-13, 8-12,9-12, etc.

In some embodiments, the internucleosidic linkages of providedoligonucleotides of such wing-core-wing (i.e., X—Y—X) motifs are allchiral, modified phosphate linkages. In some embodiments, theinternucleosidic linkages of provided oligonucleotides of suchwing-core-wing (i.e., X—Y—X) motifs are all chiral phosphorothioateinternucleotidic linkages. In some embodiments, chiral internucleotidiclinkages of provided oligonucleotides of such wing-core-wing motifs areat least about 10, 20, 30, 40, 50, 50, 70, 80, or 90% chiral, modifiedphosphate internucleotidic linkages. In some embodiments, chiralinternucleotidic linkages of provided oligonucleotides of suchwing-core-wing motifs are at least about 10, 20, 30, 40, 50, 60, 70, 80,or 90% chiral phosphorothioate internucleotidic linkages. In someembodiments, chiral internucleotidic linkages of providedoligonucleotides of such wing-core-wing motifs are at least about 10,20, 30, 40, 50, 50, 70, 80, or 90% chiral phosphorothioateinternucleotidic linkages of the Sp conformation.

In some embodiments, each wing region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif optionally contains chiral phosphorothioate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif contains chiral phosphorothioate internucleotidic linkages. Insome embodiments, the two wing regions of a wing-core-wing motif havethe same internucleotidic linkage stereochemistry. In some embodiments,the two wing regions have different internucleotidic linkagestereochemistry. In some embodiments, each internucleotidic linkage inthe wings is independently a chiral internucleotidic linkage.

In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral phosphorothioate internucleotidic linkages.In some embodiments, the core region of a wing-core-wing motif comprisesa repeating pattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif has a repeatingpattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)_(m)Rp or Rp(Sp)_(m), wherein m is 1-50. Insome embodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)_(m)Rp or Rp(Sp)_(m), wherein m is 1-50. Insome embodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)_(m)Rp, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is Rp(Sp)_(m), wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is (Sp)_(m)Rp or Rp(Sp)_(m), wherein m is 1-50. Insome embodiments, the core region of a wing-core-wing motif hasrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)_(m)Rp, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is Rp(Sp)_(m), wherein m is 1-50. In some embodiments,the core region of a wing-core-wing motif has repeating pattern ofinternucleotidic linkage stereochemistry, wherein the repeating patternis a motif comprising at least 33% of internucleotidic linkage in the Sconformation. In some embodiments, the core region of a wing-core-wingmotif has repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is a motif comprising at least 50% ofinternucleotidic linkage in the S conformation. In some embodiments, thecore region of a wing-core-wing motif has repeating pattern ofinternucleotidic linkage stereochemistry, wherein the repeating patternis a motif comprising at least 66% of internucleotidic linkage in the Sconformation. In some embodiments, the core region of a wing-core-wingmotif has repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is a repeating triplet motif selected fromRpRpSp and SpSpRp. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is a repeating RpRpSp. Insome embodiments, the core region of a wing-core-wing motif hasrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is a repeating SpSpRp.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers in the core region comprises(Sp)_(m)Rp or Rp(Sp)_(m). In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers in thecore region comprises Rp(Sp)_(m). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Sp)_(m)Rp. In some embodiments, m is 2. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises Rp(Sp)₂. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Sp)₂Rp(Sp)₂. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Rp)₂Rp(Sp)₂. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises RpSpRp(Sp)₂. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises SpRpRp(Sp)₂. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Sp)₂Rp.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)_(m)Rp or Rp(Sp)_(m).In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises Rp(Sp)_(m). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)_(m)Rp. In some embodiments, m is2. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises Rp(Sp)₂. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)₂Rp(Sp)₂. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Rp)₂Rp(Sp)₂. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises RpSpRp(Sp)₂. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers comprisesSpRpRp(Sp)₂. In some embodiments, the present disclosure provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers comprises (Sp)₂Rp.

As defined herein, m is 1-50. In some embodiments, m is 1. In someembodiments, m is 2-50. In some embodiments, m is 2, 3, 4, 5, 6, 7 or 8.In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, m is4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In someembodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8. In someembodiments, m is 2. In some embodiments, m is 3. In some embodiments, mis 4. In some embodiments, m is 5. In some embodiments, m is 6. In someembodiments, m is 7. In some embodiments, m is 8. In some embodiments, mis 9. In some embodiments, m is 10. In some embodiments, m is 11. Insome embodiments, m is 12. In some embodiments, m is 13. In someembodiments, m is 14. In some embodiments, m is 15. In some embodiments,m is 16. In some embodiments, m is 17. In some embodiments, m is 18. Insome embodiments, m is 19. In some embodiments, m is 20. In someembodiments, m is 21. In some embodiments, m is 22. In some embodiments,m is 23. In some embodiments, m is 24. In some embodiments, m is 25. Insome embodiments, m is greater than 25.

In some embodiments, a repeating pattern is (Sp)_(m)(Rp)_(n), wherein nis 1-10, and m is independently as defined above and described herein.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)_(m)(Rp)_(n). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Sp)_(m)(Rp)_(n).In some embodiments, a repeating pattern is (Rp)_(n)(Sp)_(m), wherein nis 1-10, and m is independently as defined above and described herein.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Rp)_(n)(Sp)_(m). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Rp)_(n)(Sp)_(m).In some embodiments, (Rp)_(n)(Sp)_(m) is (Rp)(Sp)₂. In some embodiments,(Sp)_(n)(Rp)_(m) is (Sp)₂(Rp).

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)_(m)(Rp)_(n)(Sp)_(t).In some embodiments, a repeating pattern is (Sp)_(m)(Rp)_(n)(Sp)_(t),wherein n is 1-10, t is 1-50, and m is as defined above and describedherein. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers in the core region comprises(Sp)_(m)(Rp)_(n)(Sp)_(t). In some embodiments, a repeating pattern is(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein n is 1-10, t is 1-50, and m is asdefined above and described herein. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Sp)_(t)(Rp)_(n)(Sp)_(m). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Sp)_(t)(Rp)_(n)(Sp)_(m).

In some embodiments, a repeating pattern is (Np)_(t)(Rp)_(n)(Sp)_(m),wherein n is 1-10, t is 1-50, Np is independently Rp or Sp, and m is asdefined above and described herein. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Np)_(t)(Rp)_(n)(Sp)_(m). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Np)_(t)(Rp)_(n)(Sp)_(m). In some embodiments, arepeating pattern is (Np)_(m)(Rp)_(n)(Sp)_(t), wherein n is 1-10, t is1-50, Np is independently Rp or Sp, and m is as defined above anddescribed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers comprises(Np)_(m)(Rp)_(n)(Sp)_(t). In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Np)_(m)(Rp)_(n)(Sp)_(t). In some embodiments, Npis Rp. In some embodiments, Np is Sp. In some embodiments, all Np arethe same. In some embodiments, all Np are Sp. In some embodiments, atleast one Np is different from the other Np. In some embodiments, t is2.

As defined herein, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5,6, 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3,4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8. In someembodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5, 6, 7 or8. In some embodiments, n is 6, 7 or 8. In some embodiments, n is 7 or8. In some embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4. In some embodiments, nis 5. In some embodiments, n is 6. In some embodiments, n is 7. In someembodiments, n is 8. In some embodiments, n is 9. In some embodiments, nis 10.

As defined herein, t is 1-50. In some embodiments, t is 1. In someembodiments, t is 2-50. In some embodiments, t is 2, 3, 4, 5, 6, 7 or 8.In some embodiments, t is 3, 4, 5, 6, 7 or 8. In some embodiments, t is4, 5, 6, 7 or 8. In some embodiments, t is 5, 6, 7 or 8. In someembodiments, t is 6, 7 or 8. In some embodiments, t is 7 or 8. In someembodiments, t is 2. In some embodiments, t is 3. In some embodiments, tis 4. In some embodiments, t is 5. In some embodiments, t is 6. In someembodiments, t is 7. In some embodiments, t is 8. In some embodiments, tis 9. In some embodiments, t is 10. In some embodiments, t is 11. Insome embodiments, t is 12. In some embodiments, t is 13. In someembodiments, t is 14. In some embodiments, t is 15. In some embodiments,t is 16. In some embodiments, t is 17. In some embodiments, t is 18. Insome embodiments, t is 19. In some embodiments, t is 20. In someembodiments, t is 21. In some embodiments, t is 22. In some embodiments,t is 23. In some embodiments, t is 24. In some embodiments, t is 25. Insome embodiments, t is greater than 25.

In some embodiments, at least one of m and t is greater than 2. In someembodiments, at least one of m and t is greater than 3. In someembodiments, at least one of m and t is greater than 4. In someembodiments, at least one of m and t is greater than 5. In someembodiments, at least one of m and t is greater than 6. In someembodiments, at least one of m and t is greater than 7. In someembodiments, at least one of m and t is greater than 8. In someembodiments, at least one of m and t is greater than 9. In someembodiments, at least one of m and t is greater than 10. In someembodiments, at least one of m and t is greater than 11. In someembodiments, at least one of m and t is greater than 12. In someembodiments, at least one of m and t is greater than 13. In someembodiments, at least one of m and t is greater than 14. In someembodiments, at least one of m and t is greater than 15. In someembodiments, at least one of m and t is greater than 16. In someembodiments, at least one of m and t is greater than 17. In someembodiments, at least one of m and t is greater than 18. In someembodiments, at least one of m and t is greater than 19. In someembodiments, at least one of m and t is greater than 20. In someembodiments, at least one of m and t is greater than 21. In someembodiments, at least one of m and t is greater than 22. In someembodiments, at least one of m and t is greater than 23. In someembodiments, at least one of m and t is greater than 24. In someembodiments, at least one of m and t is greater than 25.

In some embodiments, each one of m and t is greater than 2. In someembodiments, each one of m and t is greater than 3. In some embodiments,each one of m and t is greater than 4. In some embodiments, each one ofm and t is greater than 5. In some embodiments, each one of m and t isgreater than 6. In some embodiments, each one of m and t is greater than7. In some embodiments, each one of m and t is greater than 8. In someembodiments, each one of m and t is greater than 9. In some embodiments,each one of m and t is greater than 10. In some embodiments, each one ofm and t is greater than 11. In some embodiments, each one of m and t isgreater than 12. In some embodiments, each one of m and t is greaterthan 13. In some embodiments, each one of m and t is greater than 14. Insome embodiments, each one of m and t is greater than 15. In someembodiments, each one of m and t is greater than 16. In someembodiments, each one of m and t is greater than 17. In someembodiments, each one of m and t is greater than 18. In someembodiments, each one of m and t is greater than 19. In someembodiments, each one of m and t is greater than 20.

In some embodiments, the sum of m and t is greater than 3. In someembodiments, the sum of m and t is greater than 4. In some embodiments,the sum of m and t is greater than 5. In some embodiments, the sum of mand t is greater than 6. In some embodiments, the sum of m and t isgreater than 7. In some embodiments, the sum of m and t is greater than8. In some embodiments, the sum of m and t is greater than 9. In someembodiments, the sum of m and t is greater than 10. In some embodiments,the sum of m and t is greater than 11. In some embodiments, the sum of mand t is greater than 12. In some embodiments, the sum of m and t isgreater than 13. In some embodiments, the sum of m and t is greater than14. In some embodiments, the sum of m and t is greater than 15. In someembodiments, the sum of m and t is greater than 16. In some embodiments,the sum of m and t is greater than 17. In some embodiments, the sum of mand t is greater than 18. In some embodiments, the sum of m and t isgreater than 19. In some embodiments, the sum of m and t is greater than20. In some embodiments, the sum of m and t is greater than 21. In someembodiments, the sum of m and t is greater than 22. In some embodiments,the sum of m and t is greater than 23. In some embodiments, the sum of mand t is greater than 24. In some embodiments, the sum of m and t isgreater than 25.

In some embodiments, n is 1, and at least one of m and t is greaterthan 1. In some embodiments, n is 1 and each of m and t is independentlygreater than 1. In some embodiments, m>n and t>n. In some embodiments,(Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₂Rp(Sp)₂. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₂Rp(Sp)₂. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is SpRp(Sp)₂. In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is (Np)_(t)Rp(Sp)_(m). In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is (Np)₂Rp(Sp)_(m). In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is (Rp)₂Rp(Sp)_(m). In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₂Rp(Sp)_(m). In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is RpSpRp(Sp)_(m). In some embodiments,(Np)_(t)(Rp)_(n)(Sp)_(m) is SpRpRp(Sp)_(m).

In some embodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is SpRpSpSp. In someembodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₂Rp(Sp)₂. In someembodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₃Rp(Sp)₃. In someembodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₄Rp(Sp)₄. In someembodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)_(t)Rp(Sp)₅. In someembodiments, (Sp)_(t)(Rp)_(n)(Sp)_(m) is SpRp(Sp)₅. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₂Rp(Sp)₅. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₃Rp(Sp)₅. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₄Rp(Sp)₅. In some embodiments,(Sp)_(t)(Rp)_(n)(Sp)_(m) is (Sp)₅Rp(Sp)₅.

In some embodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₂Rp(Sp)₂. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₃Rp(Sp)₃. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₄Rp(Sp)₄. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)_(m)Rp(Sp)₅. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₂Rp(Sp)₅. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₃Rp(Sp)₅. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₄Rp(Sp)₅. In someembodiments, (Sp)_(m)(Rp)_(n)(Sp)_(t) is (Sp)₅Rp(Sp)₅.

In some embodiments, the core region comprises at least one Rpinternucleotidic linkage. In some embodiments, the core region of awing-core-wing motif comprises at least one Rp internucleotidic linkage.In some embodiments, a core region comprises at least one Rpphosphorothioate internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises at least one Rpphosphorothioate internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises only one Rp phosphorothioateinternucleotidic linkage. In some embodiments, a core region motifcomprises at least two Rp internucleotidic linkages. In someembodiments, the core region of a wing-core-wing motif comprises atleast two Rp internucleotidic linkages. In some embodiments, the coreregion of a wing-core-wing motif comprises at least two Rpphosphorothioate internucleotidic linkages. In some embodiments, a coreregion comprises at least three Rp internucleotidic linkages. In someembodiments, the core region of a wing-core-wing motif comprises atleast three Rp internucleotidic linkages. In some embodiments, the coreregion comprises at least three Rp phosphorothioate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifcomprises at least three Rp phosphorothioate internucleotidic linkages.In some embodiments, a core region comprises at least 4, 5, 6, 7, 8, 9,or 10 Rp internucleotidic linkages. In some embodiments, the core regionof a wing-core-wing motif comprises at least 4, 5, 6, 7, 8, 9, or 10 Rpinternucleotidic linkages. In some embodiments, a core region comprisesat least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifcomprises at least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioateinternucleotidic linkages.

In certain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each wing region are 2′-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OR-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-MOE-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OMe-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues in the core region are 2′-deoxyribonucleoside residues. Incertain embodiments, a wing-core-wing motif is a 5-10-5 motif, whereinall internucleotidic linkages are phosphorothioate linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif, wherein allinternucleotidic linkages are chiral phosphorothioate linkages. Incertain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each wing region are 2′-modified residues, the residuesin the core region are 2′-deoxyribonucleoside residues, and allinternucleotidic linkages in the core region are chiral phosphorothioatelinkages. In certain embodiments, a wing-core-wing motif is a 5-10-5motif wherein the residues at each wing region are 2′-OR-modifiedresidues, the residues in the core region are 2′-deoxyribonucleosideresidues, and all internucleotidic linkages in the core region arechiral phosphorothioate linkages. In certain embodiments, awing-core-wing motif is a 5-10-5 motif wherein the residues at each wingregion are 2′-MOE-modified residues, the residues in the core region are2′-deoxyribonucleoside residues, and all internucleotidic linkages inthe core region are chiral phosphorothioate linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OMe-modified residues, the residuesin the core region are 2′-deoxyribonucleoside residues, and allinternucleotidic linkages in the core region are chiral phosphorothioatelinkages.

In some embodiments, residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core motif is amotif wherein the residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a core-wing motif is amotif wherein the residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a motif wherein the residues at each “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a 5-10-5 motif wherein the residues at each “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a 5-10-5 motif wherein the residues in the core “Y” region are2′-deoxyribonucleoside residues. In certain embodiments, awing-core-wing motif is a 5-10-5 motif, wherein all internucleotidiclinkages are phosphorothioate internucleotidic linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif, wherein allinternucleotidic linkages are chiral phosphorothioate internucleotidiclinkages. In certain embodiments, a wing-core-wing motif is a 5-10-5motif wherein the residues at each “X” wing region are not2′-MOE-modified residues, the residues in the core “Y” region are2′-deoxyribonucleoside, and all internucleotidic linkages are chiralphosphorothioate internucleotidic linkages.

As understood by a person having ordinary skill in the art, providedoligonucleotides and compositions, among other things, can target agreat number of nucleic acid polymers. For instance, in someembodiments, provided oligonucleotides and compositions may target atranscript of a nucleic acid sequence, wherein a common base sequence ofoligonucleotides (e.g., a base sequence of an oligonucleotide type)comprises or is a sequence complementary to a sequence of thetranscript. In some embodiments, a common base sequence comprises asequence complimentary to a sequence of a target. In some embodiments, acommon base sequence is a sequence complimentary to a sequence of atarget. In some embodiments, a common base sequence comprises or is asequence 100% complimentary to a sequence of a target. In someembodiments, a common base sequence comprises a sequence 100%complimentary to a sequence of a target. In some embodiments, a commonbase sequence is a sequence 100% complimentary to a sequence of atarget. In some embodiments, a common base sequence in a core comprisesor is a sequence complimentary to a sequence of a target. In someembodiments, a common base sequence in a core comprises a sequencecomplimentary to a sequence of a target. In some embodiments, a commonbase sequence in a core is a sequence % complimentary to a sequence of atarget. In some embodiments, a common base sequence in a core comprisesor is a sequence 100% complimentary to a sequence of a target. In someembodiments, a common base sequence in a core comprises a sequence 100%complimentary to a sequence of a target. In some embodiments, a commonbase sequence in a core is a sequence 100% complimentary to a sequenceof a target.

In some embodiments, as described in this disclosure, providedoligonucleotides and compositions may provide new cleavage patterns,higher cleavage rate, higher cleavage degree, higher cleavageselectivity, etc. In some embodiments, provided compositions canselectively suppress (e.g., cleave) a transcript from a target nucleicacid sequence which has one or more similar sequences exist within asubject or a population, each of the target and its similar sequencescontains a specific nucleotidic characteristic sequence element thatdefines the target sequence relative to the similar sequences. In someembodiments, for example, a target sequence is a wild-type allele orcopy of a gene, and a similar sequence is a sequence has very similarbase sequence, e.g., a sequence having SNP, mutations, etc.; In someembodiments, a characteristic sequence element defines that targetsequence relative to the similar sequence: for example, when a targetsequence is a Huntington's disease-associated allele with T at rs362307(U in the corresponding RNA; C for the non-disease-associated allele), acharacteristic sequence comprises this SNP.

In some embodiments, a similar sequence has greater than 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence homology with a target sequence. In some embodiments, a targetsequence is a disease-causing copy of a nucleic acid sequence comprisingone or more mutations and/or SNPs, and a similar sequence is a copy notcausing the disease (wild type). In some embodiments, a target sequencecomprises a mutation, wherein a similar sequence is the correspondingwild-type sequence. In some embodiments, a target sequence is a mutantallele, while a similar sequence is a wild-type allele. In someembodiments, a target sequence comprises a SNP that is associated with adisease-causing allele, while a similar sequence comprises the same SNPthat is not associated with the disease-causing allele. In someembodiments, the region of a target sequence that is complementary to acommon base sequence of a provided oligonucleotide composition hasgreater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence homology with the corresponding region ofa similar sequence. In some embodiments, the region of a target sequencethat is complementary to a common base sequence of a providedoligonucleotide composition differs from the corresponding region of asimilar sequence at less than 5, less than 4, less than 3, less than 2,or only 1 base pairs. In some embodiments, the region of a targetsequence that is complementary to a common base sequence of a providedoligonucleotide composition differs from the corresponding region of asimilar sequence only at a mutation site or SNP site. In someembodiments, the region of a target sequence that is complementary to acommon base sequence of a provided oligonucleotide composition differsfrom the corresponding region of a similar sequence only at a mutationsite. In some embodiments, the region of a target sequence that iscomplementary to a common base sequence of a provided oligonucleotidecomposition differs from the corresponding region of a similar sequenceonly at a SNP site.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element. In some embodiments,a common base sequence comprises a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence is a sequence complementary to a characteristic sequenceelement. In some embodiments, a common base sequence comprises or is asequence 100% complementary to a characteristic sequence element. Insome embodiments, a common base sequence comprises a sequence 100%complementary to a characteristic sequence element. In some embodiments,a common base sequence is a sequence 100% complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core comprises or is a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core comprises a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core is a sequence complementary to a characteristicsequence element. In some embodiments, a common base sequence in a corecomprises or is a sequence 100% complementary to a characteristicsequence element. In some embodiments, a common base sequence in a corecomprises a sequence 100% complementary to a characteristic sequenceelement. In some embodiments, a common base sequence in a core is asequence 100% complementary to a characteristic sequence element.

In some embodiments, a characteristic sequence element comprises or is amutation. In some embodiments, a characteristic sequence elementcomprises a mutation. In some embodiments, a characteristic sequenceelement is a mutation. In some embodiments, a characteristic sequenceelement comprises or is a point mutation. In some embodiments, acharacteristic sequence element comprises a point mutation. In someembodiments, a characteristic sequence element is a point mutation. Insome embodiments, a characteristic sequence element comprises or is aSNP. In some embodiments, a characteristic sequence element comprises aSNP. In some embodiments, a characteristic sequence element is a SNP.

In some embodiments, a common base sequence 100% matches a targetsequence, which it does not 100% match a similar sequence of the targetsequence. For example, in some embodiments, a common base sequencematches a mutation in the disease-causing copy or allele of a targetnucleic acid sequence, but does not match a non-disease-causing copy orallele at the mutation site; in some other embodiments, a common basesequence matches a SNP in the disease-causing allele of a target nucleicacid sequence, but does not match a non-disease-causing allele at thecorresponding site. In some embodiments, a common base sequence in acore 100% matches a target sequence, which it does not 100% match asimilar sequence of the target sequence. For example, in WV-1092, itscommon base sequence (and its common base sequence in its core) matchesthe disease-associated U, but not the non-disease-associated (wild-type)C at rs362307.

Among other things, the present disclosure recognizes that a basesequence may have impact on oligonucleotide properties. In someembodiments, a base sequence may have impact on cleavage pattern of atarget when oligonucleotides having the base sequence are utilized forsuppressing a target, e.g., through a pathway involving RNase H: forexample, FIG. 33 demonstrates that structurally similar (allphosphorothioate linkages, all stereorandom) oligonucleotides havedifferent sequences may have different cleavage patterns. In someembodiments, a common base sequence of a non-stereorandomoligonucleotide compositions (e.g., certain oligonucleotide compositionsprovided in the present disclosure) is a base sequence that when appliedto a DNA oligonucleotide composition (e.g., ONT-415) or a stereorandomall-phosphorothioate oligonucleotide composition (e.g., WV-905),cleavage pattern of the DNA (DNA cleavage pattern) and/or thestereorandom all-phosphorothioate (stereorandom cleavage pattern)composition has a cleavage site within or in the vicinity of acharacteristic sequence element. In some embodiments, a cleavage sitewithin or in the vicinity is within a sequence complementary to a coreregion of a common sequence. In some embodiments, a cleavage site withinor in the vicinity is within a sequence 100% complementary to a coreregion of a common sequence.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site within or in the vicinity of a characteristic sequenceelement in its DNA cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site within acharacteristic sequence element in its DNA cleavage pattern. In someembodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of a characteristic sequence element inits DNA cleavage pattern. In some embodiments, a common base sequence isa base sequence that has a cleavage site in the vicinity of a mutationor SNP of a characteristic sequence element in its DNA cleavage pattern.In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a mutation in its DNA cleavagepattern. In some embodiments, a common base sequence is a base sequencethat has a cleavage site in the vicinity of a SNP in its DNA cleavagepattern.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site within or in the vicinity of a characteristic sequenceelement in its stereorandom cleavage pattern. In some embodiments, acommon base sequence is a base sequence that has a cleavage site withina characteristic sequence element in its stereorandom cleavage pattern.In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a characteristic sequence element inits stereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation or SNP of a characteristic sequence element in itsstereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation in its stereorandom cleavage pattern. In some embodiments, acommon base sequence is a base sequence that has a cleavage site in thevicinity of a SNP in its stereorandom cleavage pattern.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a mutation of a characteristicsequence element in its DNA and/or stereorandom cleavage pattern. Insome embodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of a mutation in its DNA and/orstereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation in its DNA cleavage pattern. In some embodiments, a cleavagesite in the vicinity of a mutation is at a mutation, i.e., a cleavagesite is at the internucleotidic linkage of a mutated nucleotide (e.g.,if a mutation is at A in the target sequence of GGGACGTCTT (SEQ ID NO:16), the cleavage is between A and C). In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 internucleotidic linkages away from a mutation, where 0 meanscleavage at the mutation site (e.g., if a mutation is at A in the targetsequence of GGGACGTCTT (SEQ ID NO: 16), the cleavage is between A and Cfor 0 internucleotidic linkage away; a cleavage site 1 internucleotidiclinkage away from the mutation is between G and A to the 5′ from themutation or between C and G to the 3′ from the mutation). In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from a mutation. Insome embodiments, a cleavage site in the vicinity is a cleavage site 0,1, 2, 3, 4, or 5 internucleotidic linkages away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away to the5′ from a mutation. In some embodiments, a cleavage site in the vicinityis a cleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away tothe 3′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidic linkagesaway from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidic linkagesaway to the 5′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidiclinkages away to the 3′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, or 3 internucleotidiclinkages away from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, or 3 internucleotidic linkagesaway to the 5′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, or 3 internucleotidic linkagesaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, or 2 internucleotidic linkagesaway from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, or 2 internucleotidic linkages away tothe 5′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, or 2 internucleotidic linkages away tothe 3′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0 or 1 internucleotidic linkage away from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 or 1 internucleotidic linkage away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 or 1 internucleotidic linkage away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 internucleotidic linkage away from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site oneinternucleotidic linkage away from a mutation. In some embodiments, acleavage site in the vicinity is a cleavage site one internucleotidiclinkage away to the 5′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site one internucleotidic linkageaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site two internucleotidic linkages away froma mutation. In some embodiments, a cleavage site in the vicinity is acleavage site two internucleotidic linkages away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site two internucleotidic linkages away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site three internucleotidic linkages away from a mutation. Insome embodiments, a cleavage site in the vicinity is a cleavage sitethree internucleotidic linkages away to the 5′ from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site threeinternucleotidic linkages away to the 3′ from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site fourinternucleotidic linkages away from a mutation. In some embodiments, acleavage site in the vicinity is a cleavage site four internucleotidiclinkages away to the 5′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site four internucleotidic linkagesaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site five internucleotidic linkages away froma mutation. In some embodiments, a cleavage site in the vicinity is acleavage site five internucleotidic linkages away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site five internucleotidic linkages away to the 3′ from amutation.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a SNP of a characteristic sequenceelement in its DNA and/or stereorandom cleavage pattern. In someembodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of a SNP in its DNA and/or stereorandomcleavage pattern. In some embodiments, a common base sequence is a basesequence that has a cleavage site in the vicinity of a SNP in its DNAcleavage pattern. In some embodiments, a cleavage site in the vicinityof a SNP is at a SNP, i.e., a cleavage site is at the internucleotidiclinkage of a nucleotide at a SNP (e.g., for the target of WV-905,G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C (SEQ ID NO: 17), which comprisesrUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 18)(rs362307 bolded), the cleavage is between the bolded rU and theunderlined rG immediately after it). In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 internucleotidic linkages away from a SNP, where 0 means cleavage ata SNP (e.g., for the target of WV-905,G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C (SEQ ID NO: 17), which comprisesrUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 18)(rs362307 bolded), the cleavage is between the bolded rU and theunderlined rG immediately after it for 0 internucleotidic linkage away;a cleavage site 1 internucleotidic linkage away from a SNP is betweenthe rG and rU to the 5′ from the SNP (underlined:rUrUrUrGrGrArArGrUrCrUrGrUrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 18)),or between rG and rC to the 3′-end of the SNP (underlined:rUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 18))). Insome embodiments, a cleavage site in the vicinity is a cleavage site 0,1, 2, 3, 4, or 5 internucleotidic linkages away from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, or 4 internucleotidic linkages away from a SNP. In some embodiments,a cleavage site in the vicinity is a cleavage site 0, 1, 2, 3, or 4internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, or 4 internucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,or 3 internucleotidic linkages away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0, 1, 2, or 3internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,or 3 internucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, or2 internucleotidic linkages away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0, 1, or 2internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, or2 internucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0 or 1internucleotidic linkage away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0 or 1 internucleotidiclinkage away to the 5′ from a SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site 0 or 1 internucleotidic linkage awayto the 3′ from a SNP. In some embodiments, a cleavage site in thevicinity is a cleavage site 0 internucleotidic linkage away from a SNP.In some embodiments, a cleavage site in the vicinity is a cleavage siteone internucleotidic linkage away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site one internucleotidiclinkage away to the 5′ from a SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site one internucleotidic linkage away tothe 3′ from a SNP. In some embodiments, a cleavage site in the vicinityis a cleavage site two internucleotidic linkages away from a SNP. Insome embodiments, a cleavage site in the vicinity is a cleavage site twointernucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site twointernucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site threeinternucleotidic linkages away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site three internucleotidiclinkages away to the 5′ from a SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site three internucleotidic linkages awayto the 3′ from a SNP. In some embodiments, a cleavage site in thevicinity is a cleavage site four internucleotidic linkages away from aSNP. In some embodiments, a cleavage site in the vicinity is a cleavagesite four internucleotidic linkages away to the 5′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site fourinternucleotidic linkages away to the 3′ from a SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site fiveinternucleotidic linkages away from a SNP. In some embodiments, acleavage site in the vicinity is a cleavage site five internucleotidiclinkages away to the 5′ from a SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site five internucleotidic linkages awayto the 3′ from a SNP. For example, FIG. 33 demonstrates thatstereorandom cleavage pattern of the WV-905 sequence has cleavage sitesat the SNP (between CUGU and GCCC), two internucleotidic linkages away(between GUCU and GUGC, and between GUGC and CCUU), threeinternucleotidic linkages away (between UGCC and CUUG); fourinternucleotidic linkages away (between GCCC and UUGU, and AAGU andCUGU), and five internucleotidic linkages away (between CCCU and UGUG).

In some embodiments, a cleavage site within or in the vicinity of acharacteristic sequence element, e.g., in the vicinity of a mutation, aSNP, etc., is a major cleavage site of a DNA and/or stereorandomcleavage pattern. In some embodiments, a cleavage site within or in thevicinity of a characteristic sequence element is a major cleavage siteof a DNA cleavage pattern. In some embodiments, a cleavage site withinor in the vicinity of a characteristic sequence element is a majorcleavage site of a stereorandom cleavage pattern. In some embodiments, acleavage site in the vicinity of a mutation is a major cleavage site ofa DNA cleavage pattern. In some embodiments, a cleavage site in thevicinity of a mutation is a major cleavage site of a stereorandomcleavage pattern. In some embodiments, a cleavage site in the vicinityof a SNP is a major cleavage site of a DNA cleavage pattern. In someembodiments, a cleavage site in the vicinity of a SNP is a majorcleavage site of a stereorandom cleavage pattern. In some embodiments, amajor cleavage site is within a sequence complementary to a core regionof a common sequence. In some embodiments, a major cleavage site iswithin a sequence 100% complementary to a core region of a commonsequence.

In some embodiments, a major cleavage site is a site having the most, orthe second, third, fourth or fifth most cleavage. In some embodiments, amajor cleavage site is a site having the most, or the second, third, orfourth most cleavage. In some embodiments, a major cleavage site is asite having the most, or the second, or third most cleavage. In someembodiments, a major cleavage site is a site having the most or thesecond most cleavage. In some embodiments, a major cleavage site is asite having the most cleavage. In some embodiments, a major cleavagesite is a site having the second most cleavage. In some embodiments, amajor cleavage site is a site having the third most cleavage. In someembodiments, a major cleavage site is a site having the fourth mostcleavage. In some embodiments, a major cleavage site is a site havingthe fifth most cleavage.

In some embodiments, a major cleavage site is a site wherein greaterthan 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of cleavage occurs.In some embodiments, a major cleavage site is a site wherein greaterthan 5% of cleavage occurs. In some embodiments, a major cleavage siteis a site wherein greater than 10% of cleavage occurs. In someembodiments, a major cleavage site is a site wherein greater than 15% ofcleavage occurs. In some embodiments, a major cleavage site is a sitewherein greater than 20% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 25% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 30% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 35% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than40% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 45% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 50% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 55% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 60% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than65% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 70% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 75% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 80% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 85% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than90% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 91% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 92% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 93% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 94% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than95% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 96% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 97% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 98% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 99% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein 100% ofcleavage occurs.

In some embodiments, a major cleavage site is a site wherein greaterthan 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of a target iscleaved. In some embodiments, a major cleavage site is a site whereingreater than 5% of a target is cleaved. In some embodiments, a majorcleavage site is a site wherein greater than 10% of a target is cleaved.In some embodiments, a major cleavage site is a site wherein greaterthan 15% of a target is cleaved. In some embodiments, a major cleavagesite is a site wherein greater than 20% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 25% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 30% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 35% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 40% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 45% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 50% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 55% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 60% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 65% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 70% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 75% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 80% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 85% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 90% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 91% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 92% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 93% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 94% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 95% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 96% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 97% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 98% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 99% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein 100% of a target is cleaved. In some embodiments, acleavage pattern may not have a major cleavage site as no site reachesan absolute cleavage threshold level.

As a person having ordinary skill in the art understands, variousmethods may be useful for generating cleavage patterns and/or identifycleavage sites, including major cleavage site. In some embodiments, anexample of such an assay is an RNase cleavage assay as described herein;for example results, see FIG. 33, FIG. 34, etc.

In some embodiments, the present disclosure recognizes location effectsof a sequence motif complementary to a characteristic sequence element.In some embodiments, the present disclosure recognizes location effectsof a sequence motif complementary to a mutation. In some embodiments,the present disclosure recognizes location effects of a sequence motifcomplementary to a SNP.

In some embodiments, position 11, 12 or 13 of a sequence as counted fromits 5′-terminus aligns with a characteristic sequence element. In someembodiments, position 11 of a sequence as counted from its 5′-terminusaligns with a characteristic sequence element. In some embodiments,position 12 of a sequence as counted from its 5′-terminus aligns with acharacteristic sequence element. In some embodiments, position 13 of asequence as counted from its 5′-terminus aligns with a characteristicsequence element. In some embodiments, position 8, 9 or 10 of a sequenceas counted from its 3′-terminus aligns with a characteristic sequenceelement. In some embodiments, position 8 of a sequence as counted fromits 3′-terminus aligns with a characteristic sequence element. In someembodiments, position 9 of a sequence as counted from its 3′-terminusaligns with a characteristic sequence element. In some embodiments,position 10 of a sequence as counted from its 3′-terminus aligns with acharacteristic sequence element. In some embodiments, position 6, 7, or8 of a core region as counted from the 5′-terminus of the core regionaligns with a characteristic sequence element. In some embodiments,position 6 of a core region as counted from the 5′-terminus of the coreregion aligns with a characteristic sequence element. In someembodiments, position 7 of a core region as counted from the 5′-terminusof the core region aligns with a characteristic sequence element. Insome embodiments, position 8 of a core region as counted from the5′-terminus of the core region aligns with a characteristic sequenceelement. In some embodiments, position 3, 4, or 5 of a core region ascounted from the 3′-terminus of the core region aligns with acharacteristic sequence element. In some embodiments, position 3 of acore region as counted from the 3′-terminus of the core region alignswith a characteristic sequence element. In some embodiments, position 4of a core region as counted from the 3′-terminus of the core regionaligns with a characteristic sequence element. In some embodiments,position 5 of a core region as counted from the 3′-terminus of the coreregion aligns with a characteristic sequence element.

In some embodiments, position 11, 12 or 13 of a sequence as counted fromits 5′-terminus aligns with a mutation. In some embodiments, position 11of a sequence as counted from its 5′-terminus aligns with a mutation. Insome embodiments, position 12 of a sequence as counted from its5′-terminus aligns with a mutation. In some embodiments, position 13 ofa sequence as counted from its 5′-terminus aligns with a mutation. Insome embodiments, position 8, 9 or 10 of a sequence as counted from its3′-terminus aligns with a mutation. In some embodiments, position 8 of asequence as counted from its 3′-terminus aligns with a mutation. In someembodiments, position 9 of a sequence as counted from its 3′-terminusaligns with a mutation. In some embodiments, position 10 of a sequenceas counted from its 3′-terminus aligns with a mutation. In someembodiments, position 6, 7, or 8 of a core region as counted from the5′-terminus of the core region aligns with a mutation. In someembodiments, position 6 of a core region as counted from the 5′-terminusof the core region aligns with a mutation. In some embodiments, position7 of a core region as counted from the 5′-terminus of the core regionaligns with a mutation. In some embodiments, position 8 of a core regionas counted from the 5′-terminus of the core region aligns with amutation. In some embodiments, position 3, 4, or 5 of a core region ascounted from the 3′-terminus of the core region aligns with a mutation.In some embodiments, position 3 of a core region as counted from the3′-terminus of the core region aligns with a mutation. In someembodiments, position 4 of a core region as counted from the 3′-terminusof the core region aligns with a mutation. In some embodiments, position5 of a core region as counted from the 3′-terminus of the core regionaligns with a mutation.

In some embodiments, position 11, 12 or 13 of a sequence as counted fromits 5′-terminus aligns with a SNP. In some embodiments, position 11 of asequence as counted from its 5′-terminus aligns with a SNP. In someembodiments, position 12 of a sequence as counted from its 5′-terminusaligns with a SNP. In some embodiments, position 13 of a sequence ascounted from its 5′-terminus aligns with a SNP. In some embodiments,position 8, 9 or 10 of a sequence as counted from its 3′-terminus alignswith a SNP. In some embodiments, position 8 of a sequence as countedfrom its 3′-terminus aligns with a SNP. In some embodiments, position 9of a sequence as counted from its 3′-terminus aligns with a SNP. In someembodiments, position 10 of a sequence as counted from its 3′-terminusaligns with a SNP. In some embodiments, position 6, 7, or 8 of a coreregion as counted from the 5′-terminus of the core region aligns with aSNP. In some embodiments, position 6 of a core region as counted fromthe 5′-terminus of the core region aligns with a SNP. In someembodiments, position 7 of a core region as counted from the 5′-terminusof the core region aligns with a SNP. In some embodiments, position 8 ofa core region as counted from the 5′-terminus of the core region alignswith a SNP. In some embodiments, position 3, 4, or 5 of a core region ascounted from the 3′-terminus of the core region aligns with a SNP. Insome embodiments, position 3 of a core region as counted from the3′-terminus of the core region aligns with a SNP. In some embodiments,position 4 of a core region as counted from the 3′-terminus of the coreregion aligns with a SNP. In some embodiments, position 5 of a coreregion as counted from the 3′-terminus of the core region aligns with aSNP.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a nucleic acid sequence. In some embodiments, a commonbase sequence comprises or is a sequence 100% complementary to a nucleicacid sequence. In some embodiments, a common base sequence comprises oris a sequence complementary to a disease-causing nucleic acid sequence.In some embodiments, a common base sequence comprises or is a sequence100% complementary to a disease-causing nucleic acid sequence. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element of disease-causingnucleic acid sequence, which characteristic sequences differentiate adisease-causing nucleic acid sequence from a non-diseasing-causingnucleic acid sequence. In some embodiments, a common base sequencecomprises or is a sequence 100% complementary to a characteristicsequence element of disease-causing nucleic acid sequence, whichcharacteristic sequences differentiate a disease-causing nucleic acidsequence from a non-diseasing-causing nucleic acid sequence. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a disease-associated nucleic acid sequence. In someembodiments, a common base sequence comprises or is a sequence 100%complementary to a disease-associated nucleic acid sequence. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element of disease-associatednucleic acid sequence, which characteristic sequences differentiate adisease-associated nucleic acid sequence from a non-diseasing-associatednucleic acid sequence. In some embodiments, a common base sequencecomprises or is a sequence 100% complementary to a characteristicsequence element of disease-associated nucleic acid sequence, whichcharacteristic sequences differentiate a disease-associated nucleic acidsequence from a non-diseasing-associated nucleic acid sequence.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a gene. In some embodiments, a common base sequencecomprises or is a sequence 100% complementary to a gene. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element of a gene, whichcharacteristic sequences differentiate the gene from a similar sequencesharing homology with the gene. In some embodiments, a common basesequence comprises or is a sequence 100% complementary to acharacteristic sequence element of a gene, which characteristicsequences differentiate the gene from a similar sequence sharinghomology with the gene. In some embodiments, a common base sequencecomprises or is a sequence complementary to characteristic sequenceelement of a target gene, which characteristic sequences comprises amutation that is not found in other copies of the gene, e.g., thewild-type copy of the gene, another mutant copy the gene, etc. In someembodiments, a common base sequence comprises or is a sequence 100%complementary to characteristic sequence element of a target gene, whichcharacteristic sequences comprises a mutation that is not found in othercopies of the gene, e.g., the wild-type copy of the gene, another mutantcopy the gene, etc.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a sequence comprising a SNP. In some embodiments, acommon base sequence comprises or is a sequence complementary to asequence comprising a SNP, and the common base sequence is 100%complementary to the SNP that is associated with a disease. For example,in some embodiments, a common base sequence is 100% complementary to aSNP associated with a Huntington's disease-associated (or -causing)allele. In some embodiments, a common base sequence is that of WV-1092,which is 100% complementary to the disease-associated allele in manyHuntington's disease patients at rs362307. In some embodiments, a SNP isrs362307. In some embodiments, a SNP is rs7685686. In some embodiments,a SNP is rs362268. In some embodiments, a SNP is rs362306. In someembodiments, a SNP is rs362331. In some embodiments, a SNP is rs2530595.In some embodiments, other example SNP site may be any of the Huntingtinsite disclosed in the present disclosure.

In some embodiments, a common base sequence comprises a sequence foundin GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 19). In some embodiments, a commonbase sequence comprises a sequence found in GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 19), wherein the sequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO:19) comprises at least 15 nucleotides. In some embodiments, a commonbase sequence is GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 19).

In some embodiments, a common base sequence comprises a sequence foundin GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 20). In some embodiments, a commonbase sequence comprises a sequence found in GAGCAGCTGCAACCTGGCAA (SEQ IDNO: 20), wherein the sequence found in GAGCAGCTGCAACCTGGCAA (SEQ ID NO:20) comprises at least 15 nucleotides. In some embodiments, a commonbase sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 20). In someembodiments, a common base sequence is GGGCACAAGGGCACAGACTT (SEQ ID NO:21). In some embodiments, a common base sequence is GAGCAGCTGCAACCTGGCAA(SEQ ID NO: 20). In some embodiments, a common base sequence isGCACAAGGGCACAGACTTCC (SEQ ID NO: 22). In some embodiments, a common basesequence is CACAAGGGCACAGACTTCCA (SEQ ID NO: 23). In some embodiments, acommon base sequence is ACAAGGGCACAGACTTCCAA (SEQ ID NO: 24). In someembodiments, a common base sequence is CAAGGGCACAGACTTCCAAA (SEQ ID NO:25). In some embodiments, a common base sequence comprises a sequencefound in GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 20). In some embodiments, acommon base sequence comprises a sequence found in GAGCAGCTGCAACCTGGCAA(SEQ ID NO: 20), wherein the sequence found in GAGCAGCTGCAACCTGGCAA (SEQID NO: 20) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 20). In someembodiments, a common base sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO:20). In some embodiments, a common base sequence is AGCAGCTGCAACCTGGCAAC(SEQ ID NO: 26). In some embodiments, a common base sequence isGCAGCTGCAACCTGGCAACA (SEQ ID NO: 27). In some embodiments, a common basesequence is CAGCTGCAACCTGGCAACAA (SEQ ID NO: 28). In some embodiments, acommon base sequence is AGCTGCAACCTGGCAACAAC (SEQ ID NO: 29). In someembodiments, a common base sequence is GCTGCAACCTGGCAACAACC (SEQ ID NO:30). In some embodiments, a common base sequence comprises a sequencefound in GGGCCAACAGCCAGCCTGCA (SEQ ID NO: 31). In some embodiments, acommon base sequence comprises a sequence found in GGGCCAACAGCCAGCCTGCA(SEQ ID NO: 31), wherein the sequence found in GGGCCAACAGCCAGCCTGCA (SEQID NO: 31) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is GGGCCAACAGCCAGCCTGCA (SEQ ID NO: 31). In someembodiments, a common base sequence is GGGCCAACAGCCAGCCTGCA (SEQ ID NO:31). In some embodiments, a common base sequence is GGCCAACAGCCAGCCTGCAG(SEQ ID NO: 32). In some embodiments, a common base sequence isGCCAACAGCCAGCCTGCAGG (SEQ ID NO: 33). In some embodiments, a common basesequence is CCAACAGCCAGCCTGCAGGA (SEQ ID NO: 34). In some embodiments, acommon base sequence is CAACAGCCAGCCTGCAGGAG (SEQ ID NO: 35). In someembodiments, a common base sequence is AACAGCCAGCCTGCAGGAGG (SEQ ID NO:36). In some embodiments, a common base sequence comprises a sequencefound in ATTAATAAATTGTCATCACC (SEQ ID NO: 37). In some embodiments, acommon base sequence comprises a sequence found in ATTAATAAATTGTCATCACC(SEQ ID NO: 37), wherein the sequence found in ATTAATAAATTGTCATCACC (SEQID NO: 37) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is ATTAATAAATTGTCATCACC (SEQ ID NO: 37). In someembodiments, a common base sequence is ATTAATAAATTGTCATCACC (SEQ ID NO:37).

In some embodiments, the present disclosure provides stereochemicaldesign parameters for oligonucleotides. That is, among other things, thepresent disclosure demonstrates impact of stereochemical structure atdifferent positions along an oligonucleotide chain, for example onstability and/or activity of the oligonucleotide, including oninteraction of the oligonucleotide with a cognate ligand and/or with aprocessing enzyme. The present disclosure specifically providesoligonucleotides whose structure incorporates or reflects the designparameters. Such oligonucleotides are new chemical entities relative tostereorandom preparations having the same base sequence and length.

In some embodiments, the present disclosure provides stereochemicaldesign parameters for antisense oligonucleotides. In some embodiments,the present disclosure specifically provides design parameter foroligonucleotides that may be bound and/or cleaved by RNaseH. In oneembodiments, the present disclosure provides stereochemical designparameters for siRNA oligonucleotides. In some embodiments, the presentdisclosure specifically provides design parameters for oligonucleotidesthat may be bound and/or cleaved by, e.g., DICER, Argonaute proteins(e.g., Argonaute-1 and Argonaute-2), etc.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral. In some embodiments, at least twoof the first, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, at least three of the first, second, third, fifth, seventh,eighth, ninth, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, at least four of the first,second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments, atleast five of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least six of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastseven of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least eight of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at least nineof the first, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, one of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkages ischiral. In some embodiments, two of the first, second, third, fifth,seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, three of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, four of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, five of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, six of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, seven of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, eight of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, nine of thefirst, second, third, fifth, seventh, eighth, ninth, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, ten of the first, second, third, fifth, seventh, eighth,ninth, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages is chiral. In some embodiments, at least two of the first,second, third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastthree of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, at least four of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral. In some embodiments, at least five of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, at least six of the first,second, third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, at leastseven of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, one of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages ischiral. In some embodiments, two of the first, second, third, fifth,seventh, eighteenth, nineteenth and twentieth internucleotidic linkagesare chiral. In some embodiments, three of the first, second, third,fifth, seventh, eighteenth, nineteenth and twentieth internucleotidiclinkages are chiral. In some embodiments, four of the first, second,third, fifth, seventh, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral. In some embodiments, five of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments, sixof the first, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages are chiral. In some embodiments,seven of the first, second, third, fifth, seventh, eighteenth,nineteenth and twentieth internucleotidic linkages are chiral. In someembodiments, eight of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages arechiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral, and at least one internucleotidiclinkage is achiral. In some embodiments, a single oligonucleotide of aprovided composition comprises a region in which at least one of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages is chiral, and at least oneinternucleotidic linkage is achiral. In some embodiments, at least twointernucleotidic linkages are achiral. In some embodiments, at leastthree internucleotidic linkages are achiral. In some embodiments, atleast four internucleotidic linkages are achiral. In some embodiments,at least five internucleotidic linkages are achiral. In someembodiments, at least six internucleotidic linkages are achiral. In someembodiments, at least seven internucleotidic linkages are achiral. Insome embodiments, at least eight internucleotidic linkages are achiral.In some embodiments, at least nine internucleotidic linkages areachiral. In some embodiments, at least 10 internucleotidic linkages areachiral. In some embodiments, at least 11 internucleotidic linkages areachiral. In some embodiments, at least 12 internucleotidic linkages areachiral. In some embodiments, at least 13 internucleotidic linkages areachiral. In some embodiments, at least 14 internucleotidic linkages areachiral. In some embodiments, at least 15 internucleotidic linkages areachiral. In some embodiments, at least 16 internucleotidic linkages areachiral. In some embodiments, at least 17 internucleotidic linkages areachiral. In some embodiments, at least 18 internucleotidic linkages areachiral. In some embodiments, at least 19 internucleotidic linkages areachiral. In some embodiments, at least 20 internucleotidic linkages areachiral. In some embodiments, one internucleotidic linkage is achiral.In some embodiments, two internucleotidic linkages are achiral. In someembodiments, three internucleotidic linkages are achiral. In someembodiments, four internucleotidic linkages are achiral. In someembodiments, five internucleotidic linkages are achiral. In someembodiments, six internucleotidic linkages are achiral. In someembodiments, seven internucleotidic linkages are achiral. In someembodiments, eight internucleotidic linkages are achiral. In someembodiments, nine internucleotidic linkages are achiral. In someembodiments, 10 internucleotidic linkages are achiral. In someembodiments, 11 internucleotidic linkages are achiral. In someembodiments, 12 internucleotidic linkages are achiral. In someembodiments, 13 internucleotidic linkages are achiral. In someembodiments, 14 internucleotidic linkages are achiral. In someembodiments, 15 internucleotidic linkages are achiral. In someembodiments, 16 internucleotidic linkages are achiral. In someembodiments, 17 internucleotidic linkages are achiral. In someembodiments, 18 internucleotidic linkages are achiral. In someembodiments, 19 internucleotidic linkages are achiral. In someembodiments, 20 internucleotidic linkages are achiral. In someembodiments, a single oligonucleotide of a provided compositioncomprises a region in which all internucleotidic linkages, except the atleast one of the first, second, third, fifth, seventh, eighth, ninth,eighteenth, nineteenth and twentieth internucleotidic linkages which ischiral, are achiral.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages is chiral, and at least one internucleotidiclinkage is phosphate. In some embodiments, a single oligonucleotide of aprovided composition comprises a region in which at least one of thefirst, second, third, fifth, seventh, eighteenth, nineteenth andtwentieth internucleotidic linkages is chiral, and at least oneinternucleotidic linkage is phosphate. In some embodiments, at least twointernucleotidic linkages are phosphate. In some embodiments, at leastthree internucleotidic linkages are phosphate. In some embodiments, atleast four internucleotidic linkages are phosphate. In some embodiments,at least five internucleotidic linkages are phosphate. In someembodiments, at least six internucleotidic linkages are phosphate. Insome embodiments, at least seven internucleotidic linkages arephosphate. In some embodiments, at least eight internucleotidic linkagesare phosphate. In some embodiments, at least nine internucleotidiclinkages are phosphate. In some embodiments, at least 10internucleotidic linkages are phosphate. In some embodiments, at least11 internucleotidic linkages are phosphate. In some embodiments, atleast 12 internucleotidic linkages are phosphate. In some embodiments,at least 13 internucleotidic linkages are phosphate. In someembodiments, at least 14 internucleotidic linkages are phosphate. Insome embodiments, at least 15 internucleotidic linkages are phosphate.In some embodiments, at least 16 internucleotidic linkages arephosphate. In some embodiments, at least 17 internucleotidic linkagesare phosphate. In some embodiments, at least 18 internucleotidiclinkages are phosphate. In some embodiments, at least 19internucleotidic linkages are phosphate. In some embodiments, at least20 internucleotidic linkages are phosphate. In some embodiments, oneinternucleotidic linkage is phosphate. In some embodiments, twointernucleotidic linkages are phosphate. In some embodiments, threeinternucleotidic linkages are phosphate. In some embodiments, fourinternucleotidic linkages are phosphate. In some embodiments, fiveinternucleotidic linkages are phosphate. In some embodiments, sixinternucleotidic linkages are phosphate. In some embodiments, seveninternucleotidic linkages are phosphate. In some embodiments, eightinternucleotidic linkages are phosphate. In some embodiments, nineinternucleotidic linkages are phosphate. In some embodiments, 10internucleotidic linkages are phosphate. In some embodiments, 11internucleotidic linkages are phosphate. In some embodiments, 12internucleotidic linkages are phosphate. In some embodiments, 13internucleotidic linkages are phosphate. In some embodiments, 14internucleotidic linkages are phosphate. In some embodiments, 15internucleotidic linkages are phosphate. In some embodiments, 16internucleotidic linkages are phosphate. In some embodiments, 17internucleotidic linkages are phosphate. In some embodiments, 18internucleotidic linkages are phosphate. In some embodiments, 19internucleotidic linkages are phosphate. In some embodiments, 20internucleotidic linkages are phosphate. In some embodiments, a singleoligonucleotide of a provided composition comprises a region in whichall internucleotidic linkages, except the at least one of the first,second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth andtwentieth internucleotidic linkages which is chiral, are phosphate.

In some embodiments, a single oligonucleotide of a provided compositioncomprises a region in which at least one of the first, second, third,fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentiethinternucleotidic linkages are chiral, and at least 10% of all theinternucleotidic linkages in the region is achiral. In some embodiments,a single oligonucleotide of a provided composition comprises a region inwhich at least one of the first, second, third, fifth, seventh,eighteenth, nineteenth and twentieth internucleotidic linkages ischiral, and at least 10% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 20% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 30% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 40% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 50% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 60% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 70% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 80% of all theinternucleotidic linkages in the region are achiral. In someembodiments, at least 90% of all the internucleotidic linkages in theregion are achiral. In some embodiments, at least 50% of all theinternucleotidic linkages in the region are achiral. In someembodiments, an achiral internucleotidic linkage is a phosphate linkage.In some embodiments, each achiral internucleotidic linkage in aphosphate linkage.

In some embodiments, the first internucleotidic linkage of the region isan Sp modified internucleotidic linkage. In some embodiments, the firstinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the secondinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the secondinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the thirdinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the thirdinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the fifthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the fifthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the seventhinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the seventhinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the eighthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the eighthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the ninthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the ninthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the eighteenthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the eighteenthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the nineteenthinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the nineteenthinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage. In some embodiments, the twentiethinternucleotidic linkage of the region is an Sp modifiedinternucleotidic linkage. In some embodiments, the twentiethinternucleotidic linkage of the region is an Rp modifiedinternucleotidic linkage.

In some embodiments, the region has a length of at least 21 bases. Insome embodiments, the region has a length of 21 bases. In someembodiments, a single oligonucleotide in a provided composition has alength of at least 21 bases. In some embodiments, a singleoligonucleotide in a provided composition has a length of 21 bases.

In some embodiments, a chiral internucleotidic linkage has the structureof formula I. In some embodiments, a chiral internucleotidic linkage isphosphorothioate. In some embodiments, each chiral internucleotidiclinkage in a single oligonucleotide of a provided compositionindependently has the structure of formula I. In some embodiments, eachchiral internucleotidic linkage in a single oligonucleotide of aprovided composition is a phosphorothioate.

In some embodiments, oligonucleotides of the present disclosure compriseone or more modified sugar moieties. In some embodiments,oligonucleotides of the present disclosure comprise one or more modifiedbase moieties. As known by a person of ordinary skill in the art anddescribed in the disclosure, various modifications can be introduced toa sugar and/or moiety. For example, in some embodiments, a modificationis a modification described in U.S. Pat. No. 9,006,198 andWO2014/012081, the sugar and base modifications of each of which areincorporated herein by reference.

In some embodiments, a sugar modification is a 2′-modification. Commonlyused 2′-modifications include but are not limited to 2′-OR¹, wherein R¹is not hydrogen. In some embodiments, a modification is 2′-OR, wherein Ris optionally substituted aliphatic. In some embodiments, a modificationis 2′-OMe. In some embodiments, a modification is 2′-MOE. In someembodiments, the present disclosure demonstrates that inclusion and/orlocation of particular chirally pure internucleotidic linkages canprovide stability improvements comparable to or better than thoseachieved through use of modified backbone linkages, bases, and/orsugars. In some embodiments, a provided single oligonucleotide of aprovided composition has no modifications on the sugars. In someembodiments, a provided single oligonucleotide of a provided compositionhas no modifications on 2′-positions of the sugars (i.e., the two groupsat the 2′-position are either —H/—H or —H/—OH). In some embodiments, aprovided single oligonucleotide of a provided composition does not haveany 2′-MOE modifications.

In some embodiments, a 2′-modification is —O-L- or -L- which connectsthe 2′-carbon of a sugar moiety to another carbon of a sugar moiety. Insome embodiments, a 2′-modification is —O-L- or -L- which connects the2′-carbon of a sugar moiety to the 4′-carbon of a sugar moiety. In someembodiments, a 2′-modification is S-cEt. In some embodiments, a modifiedsugar moiety is an LNA moiety.

In some embodiments, a 2′-modification is —F. In some embodiments, a2′-modification is FANA. In some embodiments, a 2′-modification is FRNA.

In some embodiments, a sugar modification is a 5′-modification, e.g.,R-5′-Me, S-5′-Me, etc.

In some embodiments, a sugar modification changes the size of the sugarring. In some embodiments, a sugar modification is the sugar moiety inFHNA.

In some embodiments, a single oligonucleotide in a provided compositionis a better substrate for Argonaute proteins (e.g., hAgo-1 and hAgo-2)compared to stereorandom oligonucleotide compositions. Selection and/orlocation of chirally pure linkages as described in the present closureare useful design parameters for oligonucleotides that interacting withsuch proteins, such as siRNA.

In some embodiments, a single oligonucleotide in a provided compositionhas at least about 25% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 30% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 35% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 40%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 45% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 50% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 55% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 60% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 65% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 70%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 75% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 80% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 85% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 90% of its internucleotidiclinkages in Sp configuration.

In some embodiments, oligonucleotides in a provided composition is notan oligonucleotide selected from: T_(k)T_(k)^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 38),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methylcytosine modified nucleoside, and allinternucleoside linkages are phosphorothioates (PS) with stereochemistrypatterns selected from RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR,RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS,SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from: T_(k)T_(k)^(m)C_(k)AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 39),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methylcytosine modified nucleoside and allcore internucleoside linkages are phosphorothioates (PS) withstereochemistry patterns selected from: RSSSRSRRRS, RSSSSSSSSS,SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS,SSRRSSRSRS, SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR,RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR,RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS,RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR andSSSSSSSSSS.

Chirally Controlled Oligonucleotides and Chirally ControlledOligonucleotide Compositions

The present disclosure provides chirally controlled oligonucleotides,and chirally controlled oligonucleotide compositions which are of highcrude purity and of high diastereomeric purity. In some embodiments, thepresent disclosure provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity. In some embodiments, the present disclosure provides chirallycontrolled oligonucleotides, and chirally controlled oligonucleotidecompositions which are of high diastereomeric purity.

In some embodiments, a chirally controlled oligonucleotide compositionis a substantially pure preparation of an oligonucleotide type in thatoligonucleotides in the composition that are not of the oligonucleotidetype are impurities form the preparation process of said oligonucleotidetype, in some case, after certain purification procedures.

In some embodiments, the present disclosure provides oligonucleotidescomprising one or more diastereomerically pure internucleotidic linkageswith respect to the chiral linkage phosphorus. In some embodiments, thepresent disclosure provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I. In some embodiments, the present disclosure providesoligonucleotides comprising one or more diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus,and one or more phosphate diester linkages. In some embodiments, thepresent disclosure provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I, and one or more phosphate diester linkages. In someembodiments, the present disclosure provides oligonucleotides comprisingone or more diastereomerically pure internucleotidic linkages having thestructure of formula I-c, and one or more phosphate diester linkages. Insome embodiments, such oligonucleotides are prepared by usingstereoselective oligonucleotide synthesis, as described in thisapplication, to form pre-designed diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus.For instance, in one example oligonucleotide of (Rp/Sp, Rp/Sp, Rp/Sp,Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGs1Cs1As1CsC] (SEQ ID NO: 40), thefirst three internucleotidic linkages are constructed using traditionaloligonucleotide synthesis method, and the diastereomerically pureinternucleotidic linkages are constructed with stereochemical control asdescribed in this application. Example internucleotidic linkages,including those having structures of formula I, are further describedbelow.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In certain embodiments, the present disclosure provides achirally controlled oligonucleotide, wherein at least two individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another. In certain embodiments, thepresent disclosure provides a chirally controlled oligonucleotide,wherein at least two of the individual internucleotidic linkages withinthe oligonucleotide have different P-modifications relative to oneanother, and wherein the chirally controlled oligonucleotide comprisesat least one phosphate diester internucleotidic linkage. In certainembodiments, the present disclosure provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate diesterinternucleotidic linkage. In certain embodiments, the present disclosureprovides a chirally controlled oligonucleotide, wherein at least two ofthe individual internucleotidic linkages within the oligonucleotide havedifferent P-modifications relative to one another, and wherein thechirally controlled oligonucleotide comprises at least onephosphorothioate triester internucleotidic linkage. In certainembodiments, the present disclosure provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterinternucleotidic linkage.

In certain embodiments, a modified internucleotidic linkages has thestructure of formula I:

wherein each variable is as defined and described below. In someembodiments, a linkage of formula I is chiral. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula I.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent P-modifications relative to one another. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula I,and wherein individual internucleotidic linkages of formula I within theoligonucleotide have different —X-L-R¹ relative to one another. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different X relative to oneanother. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent -L-R¹ relative to one another. In some embodiments, a chirallycontrolled oligonucleotide is an oligonucleotide in a providedcomposition that is of the particular oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide is an oligonucleotidein a provided composition that has the common base sequence and length,the common pattern of backbone linkages, and the common pattern ofbackbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry relative to one another, and wherein at least a portionof the structure of the chirally controlled oligonucleotide ischaracterized by a repeating pattern of alternating stereochemistry.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another, in that they have different Xatoms in their —XLR¹ moieties, and/or in that they have different Lgroups in their —XLR moieties, and/or that they have different R¹ atomsin their —XLR¹ moieties.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to one anotherand the oligonucleotide has a structure represented by the followingformula:

[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny]

wherein:each R^(B) independently represents a block of nucleotide units havingthe R configuration at the linkage phosphorus;each S^(B) independently represents a block of nucleotide units havingthe S configuration at the linkage phosphorus;each of n1-ny is zero or an integer, with the requirement that at leastone odd n and at least one even n must be non-zero so that theoligonucleotide includes at least two individual internucleotidiclinkages with different stereochemistry relative to one another; andwherein the sum of n1-ny is between 2 and 200, and in some embodimentsis between a lower limit selected from the group consisting of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more and an upper limit selected from the group consisting of5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upperlimit being larger than the lower limit.

In some such embodiments, each n has the same value; in someembodiments, each even n has the same value as each other even n; insome embodiments, each odd n has the same value each other odd n; insome embodiments, at least two even ns have different values from oneanother; in some embodiments, at least two odd ns have different valuesfrom one another.

In some embodiments, at least two adjacent ns are equal to one another,so that a provided oligonucleotide includes adjacent blocks of Sstereochemistry linkages and R stereochemistry linkages of equallengths. In some embodiments, provided oligonucleotides includerepeating blocks of S and R stereochemistry linkages of equal lengths.In some embodiments, provided oligonucleotides include repeating blocksof S and R stereochemistry linkages, where at least two such blocks areof different lengths from one another; in some such embodiments each Sstereochemistry block is of the same length, and is of a differentlength from each R stereochemistry length, which may optionally be ofthe same length as one another.

In some embodiments, at least two skip-adjacent ns are equal to oneanother, so that a provided oligonucleotide includes at least two blocksof linkages of a first stereochemistry that are equal in length to oneanother and are separated by a block of linkages of the otherstereochemistry, which separating block may be of the same length or adifferent length from the blocks of first stereochemistry.

In some embodiments, ns associated with linkage blocks at the ends of aprovided oligonucleotide are of the same length. In some embodiments,provided oligonucleotides have terminal blocks of the same linkagestereochemistry. In some such embodiments, the terminal blocks areseparated from one another by a middle block of the other linkagestereochemistry.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoblockmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoskipmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoaltmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a gapmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is of any of theabove described patterns and further comprises patterns ofP-modifications. For instance, in some embodiments, a providedoligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoskipmer and P-modification skipmer. Insome embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] and is astereoblockmer and P-modification altmer. In some embodiments, aprovided oligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoaltmer and P-modification blockmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a chirallycontrolled oligonucleotide comprising one or more modifiedinternuceotidic linkages independently having the structure of formulaI:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene,    —C≡—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—,    —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—, —C(O)S—, —OC(O)—, and    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted group selected from C₁-C₆    alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,    —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,    —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,    —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—,    —C(O)S—, —OC(O)—, and —C(O)O—-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, and    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, and    heterocyclyl; and-   each

-    independently represents a connection to a nucleoside.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—;

-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—; each R′ is    independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each

-    independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises oneor more modified internucleotidic phosphorus linkages. In someembodiments, a chirally controlled oligonucleotide comprises, e.g., aphosphorothioate or a phosphorothioate triester linkage. In someembodiments, a chirally controlled oligonucleotide comprises aphosphorothioate triester linkage. In some embodiments, a chirallycontrolled oligonucleotide comprises at least two phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least three phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least four phosphorothioate triester linkages. In someembodiments, a chirally controlled oligonucleotide comprises at leastfive phosphorothioate triester linkages. Examples of such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a chirally controlled oligonucleotide comprisesdifferent internucleotidic phosphorus linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least one modifiedinternucleotidic linkage. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast two phosphorothioate triester linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast five phosphorothioate triester linkages. Examples of such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a phosphorothioate triester linkage comprises achiral auxiliary, which, for example, is used to control thestereoselectivity of a reaction. In some embodiments, a phosphorothioatetriester linkage does not comprise a chiral auxiliary. In someembodiments, a phosphorothioate triester linkage is intentionallymaintained until and/or during the administration to a subject.

In some embodiments, a chirally controlled oligonucleotide is linked toa solid support. In some embodiments, a chirally controlledoligonucleotide is cleaved from a solid support.

In some embodiments, a chirally controlled oligonucleotide comprises atleast one phosphate diester internucleotidic linkage and at least twoconsecutive modified internucleotidic linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least two consecutivephosphorothioate triester internucleotidic linkages.

In some embodiments, a chirally controlled oligonucleotide is ablockmer. In some embodiments, a chirally controlled oligonucleotide isa stereoblockmer. In some embodiments, a chirally controlledoligonucleotide is a P-modification blockmer. In some embodiments, achirally controlled oligonucleotide is a linkage blockmer.

In some embodiments, a chirally controlled oligonucleotide is an altmer.In some embodiments, a chirally controlled oligonucleotide is astereoaltmer. In some embodiments, a chirally controlled oligonucleotideis a P-modification altmer. In some embodiments, a chirally controlledoligonucleotide is a linkage altmer.

In some embodiments, a chirally controlled oligonucleotide is a unimer.In some embodiments, a chirally controlled oligonucleotide is astereounimer. In some embodiments, a chirally controlled oligonucleotideis a P-modification unimer. In some embodiments, a chirally controlledoligonucleotide is a linkage unimer.

In some embodiments, a chirally controlled oligonucleotide is a gapmer.

In some embodiments, a chirally controlled oligonucleotide is a skipmer.

In some embodiments, the present disclosure provides oligonucleotidescomprising one or more modified internucleotidic linkages independentlyhaving the structure of formula I:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene,    —C≡—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—,    —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—, —C(O)S—, —OC(O)—, and    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted group selected from C₁-C₆    alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,    —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,    —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,    —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—,    —C(O)S—, —OC(O)—, and —C(O)O—-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, and    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, and    heterocyclyl; and-   each

-    independently represents a connection to a nucleoside.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—;

-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each

-    independently represents a connection to a nucleoside.

In some embodiments, P* is an asymmetric phosphorus atom and is eitherRp or Sp. In some embodiments, P* is Rp. In other embodiments, P* is Sp.In some embodiments, an oligonucleotide comprises one or moreinternucleotidic linkages of formula I wherein each P* is independentlyRp or Sp. In some embodiments, an oligonucleotide comprises one or moreinternucleotidic linkages of formula I wherein each P* is Rp. In someembodiments, an oligonucleotide comprises one or more internucleotidiclinkages of formula I wherein each P* is Sp. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein P* is Rp. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein P*is Sp. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein P* is Rp, and at least oneinternucleotidic linkage of formula I wherein P* is Sp.

In some embodiments, W is O, S, or Se. In some embodiments, W is O. Insome embodiments, W is S. In some embodiments, W is Se. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein W is O. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein W is S. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Wis Se.

In some embodiments, each R is independently hydrogen, or an optionallysubstituted group selected from C₁-C₆ aliphatic, phenyl, carbocyclyl,aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is hydrogen. In some embodiments, R is anoptionally substituted group selected from C₁-C₆ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R is an optionally substituted C₁-C₆ alkyl. In someembodiments, R is optionally substituted, linear or branched hexyl. Insome embodiments, R is optionally substituted, linear or branchedpentyl. In some embodiments, R is optionally substituted, linear orbranched butyl. In some embodiments, R is optionally substituted, linearor branched propyl. In some embodiments, R is optionally substitutedethyl. In some embodiments, R is optionally substituted methyl.

In some embodiments, R is optionally substituted phenyl. In someembodiments, R is substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is optionally substituted carbocyclyl. In someembodiments, R is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R is optionally substituted monocyclic carbocyclyl. In someembodiments, R is optionally substituted cycloheptyl. In someembodiments, R is optionally substituted cyclohexyl. In someembodiments, R is optionally substituted cyclopentyl. In someembodiments, R is optionally substituted cyclobutyl. In someembodiments, R is an optionally substituted cyclopropyl. In someembodiments, R is optionally substituted bicyclic carbocyclyl.

In some embodiments, R is an optionally substituted aryl. In someembodiments, R is an optionally substituted bicyclic aryl ring.

In some embodiments, R is an optionally substituted heteroaryl. In someembodiments, R is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R is a substituted 5-6membered monocyclic heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Example R groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Example Rgroups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having 1 heteroatom independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted indolyl. In some embodiments, R is an optionally substitutedazabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted azaindolyl. In some embodiments, R is anoptionally substituted benzimidazolyl. In some embodiments, R is anoptionally substituted benzothiazolyl. In some embodiments, R is anoptionally substituted benzoxazolyl. In some embodiments, R is anoptionally substituted indazolyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is an optionally substituted quinolinyl.In some embodiments, R is an optionally substituted isoquinolinyl.According to one aspect, R is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is a quinazoline ora quinoxaline.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 6 membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atom.

In certain embodiments, R is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, Ris an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is an optionally substituted indolinyl. In someembodiments, R is an optionally substituted isoindolinyl. In someembodiments, R is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, each R′ is independently —R, —C(O)R, —CO₂R, or—SO₂R, or:

-   -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring.

In some embodiments, R′ is —R, —C(O)R, —CO₂R, or —SO₂R, wherein R is asdefined above and described herein.

In some embodiments, R′ is —R, wherein R is as defined and describedabove and herein. In some embodiments, R′ is hydrogen.

In some embodiments, R′ is —C(O)R, wherein R is as defined above anddescribed herein. In some embodiments, R′ is —CO₂R, wherein R is asdefined above and described herein. In some embodiments, R′ is —SO₂R,wherein R is as defined above and described herein.

In some embodiments, two R′ on the same nitrogen are taken together withtheir intervening atoms to form an optionally substituted heterocyclicor heteroaryl ring. In some embodiments, two R′ on the same carbon aretaken together with their intervening atoms to form an optionallysubstituted aryl, carbocyclic, heterocyclic, or heteroaryl ring.

In some embodiments, -Cy- is an optionally substituted bivalent ringselected from phenylene, carbocyclylene, arylene, heteroarylene, orheterocyclylene.

In some embodiments, -Cy- is optionally substituted phenylene. In someembodiments, -Cy- is optionally substituted carbocyclylene. In someembodiments, -Cy- is optionally substituted arylene. In someembodiments, -Cy- is optionally substituted heteroarylene. In someembodiments, -Cy- is optionally substituted heterocyclylene.

In some embodiments, each of X, Y and Z is independently —O—, —S—,—N(-L-R¹)—, or L, wherein each of L and R¹ is independently as definedabove and described below.

In some embodiments, X is —O—. In some embodiments, X is —S—. In someembodiments, X is —O— or —S—. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Xis —O—. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein X is —S—. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein X is —O—, and at least one internucleotidiclinkage of formula I wherein X is —S—. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein X is —O—, and at least one internucleotidic linkage offormula I wherein X is —S—, and at least one internucleotidic linkage offormula I wherein L is an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, X is —N(-L-R¹)—. In some embodiments, X is —N(R¹)—.In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R)—. Insome embodiments, X is —NH—.

In some embodiments, X is L. In some embodiments, X is a covalent bond.In some embodiments, X is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, X is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is —N(-L-R¹)—. In some embodiments, Y is —N(R¹)—.In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R)—. Insome embodiments, Y is —NH—.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond.In some embodiments, Y is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Y is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is —N(-L-R¹)—. In some embodiments, Z is —N(R¹)—.In some embodiments, Z is —N(R′)—. In some embodiments, Z is —N(R)—. Insome embodiments, Z is —NH—.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond.In some embodiments, Z is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Z is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Z is methylene.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L has the structure of -L¹-V—, wherein: L¹ is anoptionally substituted group selected from

C₁-C₆ alkylene, C₁-C₆ alkenylene, carbocyclylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;V is selected from —O—, —S—, —NR′—, C(R′)₂, —S—S—, —B—S—S—C—,

or an optionally substituted group selected from C₁-C₆ alkylene,arylene, C₁-C₆ heteroalkylene, heterocyclylene, and heteroarylene;

A is ═O, ═S, ═NR′, or ═C(R′)₂;

each of B and C is independently —O—, —S—, —NR′—, —C(R′)₂—, or anoptionally substituted group selected from C₁-C₆ alkylene,carbocyclylene, arylene, heterocyclylene, or heteroarylene; and each R′is independently as defined above and described herein.

In some embodiments, L¹ is

In some embodiments, L¹ is

wherein Ring Cy′ is an optionally substituted arylene, carbocyclylene,heteroarylene, or heterocyclylene. In some embodiments, L¹ is optionallysubstituted

In some embodiments, L¹ is

In some embodiments, L¹ is connected to X. In some embodiments, L¹ is anoptionally substituted group selected from

and the sulfur atom is connect to V. In some embodiments, L¹ is anoptionally substituted group selected from

and the carbon atom is connect to X.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, carbocyclic,    heteroaryl or heterocyclic ring; and each R′ is independently as    defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond; and    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

-   wherein:-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;    the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein:

-   is a single or double bond; and-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond; and-   the two R^(L)1 are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, E is —O—, —S—, —NR′— or —C(R′)₂—, wherein each R′independently as defined above and described herein. In someembodiments, E is —O—, —S—, or —NR′—. In some embodiments, E is —O—,—S—, or —NH—. In some embodiments, E is —O—. In some embodiments, E is—S—. In some embodiments, E is —NH—.

In some embodiments, G is —O—, —S—, or —NR′, wherein each R′independently as defined above and described herein. In someembodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. Insome embodiments, G is —S—. In some embodiments, G is —NH—.

In some embodiments, L is -L³-G-, wherein:

-   L³ is an optionally substituted C₁-C₅ alkylene or alkenylene,    wherein one or more methylene units are optionally and independently    replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—,    —S(O)₂—, or

-    and    wherein each of G, R′ and Ring Cy′ is independently as defined above    and described herein.

In some embodiments, L is -L³-S—, wherein L³ is as defined above anddescribed herein. In some embodiments, L is -L³-O—, wherein L³ is asdefined above and described herein. In some embodiments, L is-L³-N(R′)—, wherein each of L³ and R′ is independently as defined aboveand described herein. In some embodiments, L is -L³-NH—, wherein each ofL³ and R′ is independently as defined above and described herein.

In some embodiments, L³ is an optionally substituted Cs alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Ring Cy′ is independently as defined above anddescribed herein. In some embodiments, L³ is an optionally substitutedC₅ alkylene. In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₄ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₃ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L³ is an optionally substituted C₂ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

wherein each of G and Cy′ is independently as defined above anddescribed herein. In some embodiments, L is

In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; and G is as defined above and describedherein. In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; G is as defined above and described herein;and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted methylene; G is as defined above anddescribed herein; and G is connected to R¹. In some embodiments, L is-L⁴-G-, wherein L⁴ is methylene; G is as defined above and describedherein; and G is connected to R¹. In some embodiments, L is -L⁴-G-,wherein L⁴ is an optionally substituted —(CH₂)₂—; G is as defined aboveand described herein; and G is connected to R¹. In some embodiments, Lis -L⁴-G-, wherein L⁴ is —(CH₂)₂—; G is as defined above and describedherein; and G is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein the sulfur atom is connected to R¹. In some embodiments, L is

wherein the oxygen atom is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein.

In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3)is an optionally substituted, linear or branched, C₁-C₉ alkylene,wherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -Cy- isindependently as defined above and described herein. In someembodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is anoptionally substituted C₁-C₆ alkylene. In some embodiments, L is—S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkenylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkylene wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkenylene,arylene, or heteroarylene. In some embodiments, In some embodiments,R^(L3) is an optionally substituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆alkylene)-, —S—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-,—S—CO-arylene-(C₁-C₆ alkylene)-, or —S—CO—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-.

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments,

In some embodiments, the sulfur atom in the L embodiments describedabove and herein is connected to X. In some embodiments, the sulfur atomin the L embodiments described above and herein is connected to R¹.

In some embodiments, R¹ is halogen, R, or an optionally substitutedC₁-C₅₀ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is halogen, R, or an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is halogen.In some embodiments, R¹ is —F. In some embodiments, R¹ is —Cl. In someembodiments, R¹ is —Br. In some embodiments, R¹ is —I.

In some embodiments, R¹ is R wherein R is as defined above and describedherein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is anoptionally substituted group selected from C₁-C₅₀ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R¹ is an optionally substituted C₁-C₆ alkyl. In someembodiments, R¹ is optionally substituted, linear or branched hexyl. Insome embodiments, R¹ is optionally substituted, linear or branchedpentyl. In some embodiments, R¹ is optionally substituted, linear orbranched butyl. In some embodiments, R¹ is optionally substituted,linear or branched propyl. In some embodiments, R¹ is optionallysubstituted ethyl. In some embodiments, R¹ is optionally substitutedmethyl.

In some embodiments, R¹ is optionally substituted phenyl. In someembodiments, R¹ is substituted phenyl. In some embodiments, R¹ isphenyl.

In some embodiments, R¹ is optionally substituted carbocyclyl. In someembodiments, R¹ is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R¹ is optionally substituted monocyclic carbocyclyl. Insome embodiments, R¹ is optionally substituted cycloheptyl. In someembodiments, R¹ is optionally substituted cyclohexyl. In someembodiments, R¹ is optionally substituted cyclopentyl. In someembodiments, R¹ is optionally substituted cyclobutyl. In someembodiments, R¹ is an optionally substituted cyclopropyl. In someembodiments, R¹ is optionally substituted bicyclic carbocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ polycyclichydrocarbon. In some embodiments, R¹ is an optionally substituted C₁-C₅₀polycyclic hydrocarbon wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein eachvariable is independently as defined above and described herein. In someembodiments, R¹ is optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is optionally substituted

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphaticcomprising one or more optionally substituted polycyclic hydrocarbonmoieties. In some embodiments, R¹ is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclichydrocarbon moieties, wherein one or more methylene units are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic comprisingone or more optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted aryl. In someembodiments, R¹ is an optionally substituted bicyclic aryl ring.

In some embodiments, R¹ is an optionally substituted heteroaryl. In someembodiments, R¹ is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R¹ is a substituted5-6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an unsubstituted 5-6 membered monocyclic heteroarylring having 1-3 heteroatoms independently selected from nitrogen,sulfur, or oxygen.

In some embodiments, R¹ is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R¹ is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R¹ is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R¹ is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Example R¹ groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R¹ is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R¹ is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R¹ is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R¹ is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Example R¹groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R¹ is an optionally substituted5,6-fused heteroaryl ring having 1 heteroatom independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted indolyl. In some embodiments, R¹ is an optionallysubstituted azabicyclo[3.2.1]octanyl. In certain embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted azaindolyl. In someembodiments, R¹ is an optionally substituted benzimidazolyl. In someembodiments, R¹ is an optionally substituted benzothiazolyl. In someembodiments, R¹ is an optionally substituted benzoxazolyl. In someembodiments, R¹ is an optionally substituted indazolyl. In certainembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 3 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In certain embodiments, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R¹ is an optionally substituted quinolinyl.In some embodiments, R¹ is an optionally substituted isoquinolinyl.According to one aspect, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is a quinazoline ora quinoxaline.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 6 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atoms.

In certain embodiments, R¹ is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹ isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R¹is an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹ is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R¹ is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R¹ is an optionally substituted indolinyl. In someembodiments, R¹ is an optionally substituted isoindolinyl. In someembodiments, R¹ is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R¹ is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently asdefined above and described herein. In some embodiments, R¹ is anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionally-Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —OC(O)—, or —C(O)O—, wherein eachR′ is independently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —OC(O)—, or—C(O)O—, wherein each R′ is independently as defined above and describedherein.

In some embodiments, R¹ is

In some embodiments, R¹ is CH₃—,

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to L. Examples of such R¹ groups aredepicted below:

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to L. Example such R¹ groups aredepicted below:

In some embodiments, R¹ is —S—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —S—R^(L2), wherein the sulfur atom is connected withthe sulfur atom in L group.

In some embodiments, R¹ is —C(O)—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —C(O)—R^(L2), wherein the carbonyl group is connectedwith G in L group. In some embodiments, R¹ is —C(O)—R^(L2), wherein thecarbonyl group is connected with the sulfur atom in L group.

In some embodiments, R^(L2) is optionally substituted C₁-C₉ aliphatic.In some embodiments, R^(L2) is optionally substituted C₁-C₉ alkyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkenyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkynyl. Insome embodiments, R^(L2) is an optionally substituted C₁-C₉ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by -Cy- or —C(O)—. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by -Cy-. In some embodiments,R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted heterocycylene. In some embodiments, R^(L2) is anoptionally substituted C₁-C₉ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted arylene. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substitutedheteroarylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₃-C₁₀carbocyclylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein two methylene units are optionally andindependently replaced by -Cy- or —C(O)—. In some embodiments, R^(L2) isan optionally substituted C₁-C₉ aliphatic wherein two methylene unitsare optionally and independently replaced by -Cy- or —C(O)—. ExampleR^(L2) groups are depicted below:

In some embodiments, R¹ is hydrogen, or an optionally substituted groupselected from

—S—(C₁-C₁₀ aliphatic), C₁-C₁₀ aliphatic, aryl, C₁-C₆ heteroalkyl,heteroaryl and heterocyclyl. In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic). In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted group selected from—S—(C₁-C₆ aliphatic), C₁-C₁₀ aliphatic, C₁-C₆ heteroaliphatic, aryl,heterocyclyl and heteroaryl.

In some embodiments, R¹ is

In some embodiments, the sulfur atom in the R¹ embodiments describedabove and herein is connected with the sulfur atom, G, E, or —C(O)—moiety in the L embodiments described above and herein. In someembodiments, the —C(O)— moiety in the R¹ embodiments described above andherein is connected with the sulfur atom, G, E, or —C(O)— moiety in theL embodiments described above and herein.

In some embodiments, -L-R¹ is any combination of the L embodiments andR¹ embodiments described above and herein.

In some embodiments, -L-R¹ is -L³-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L⁴-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-C(O)—R^(L2), wherein each variableis independently as defined above and described herein.

In some embodiments, -L-R¹ is

wherein R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—, and each G is independently as defined above and describedherein.

In some embodiments, -L-R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, L has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —X-L-R¹ has the structure of:

wherein:the phenyl ring is optionally substituted, andeach of R¹ and X is independently as defined above and described herein.

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is:

In some embodiments, -L-R¹ is CH₃—,

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)₂— moiety which is connected to X. Examplesof such -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)— moiety which is connected to X. Examples ofsuch -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is CH₃—,

and X is —S—.

In some embodiments, -L-R¹ is CH₃—,

X is —S—, W is O, Y is —O—, and Z is —O—.

In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, R¹ is

In some embodiments, X is —O— or —S—, and R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, X is —O— or —S—, and R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, L is a covalent bond and -L-R¹ is R¹.

In some embodiments, -L-R¹ is not hydrogen.

In some embodiments, —X-L-R¹ is R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, —X-L-R¹ has the structure of

wherein the

moiety is optionally substituted. In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, Y′ is —O—, —S— or —NR′—, and the

moiety is optionally substituted. In some embodiments, Y′ is —O—, —S— or—NH—. In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, and the

moiety is optionally substituted. In some embodiments,

In some embodiments, —X-L-R¹ is

wherein the

is optionally substituted. In some embodiments, —X-L-R¹ is

wherein the

is substituted. In some embodiments, —X-L-R¹ is

wherein the

is unsubstituted.

In some embodiments, —X-L-R¹ is R¹—C(O)—S-L^(x)-S—, wherein L^(x) is anoptionally substituted group selected from

In some embodiments, L^(x) is

In some embodiments, —X-L-R¹ is (CH₃)₃C—S—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R′—C(═X′)—Y′—C(R)₂—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R—C(═X′)—Y′—CH₂—S-L^(x)-S—. In some embodiments,—X-L-R¹ is

As will be appreciated by a person skilled in the art, many of the—X-L-R¹ groups described herein are cleavable and can be converted to—X⁻ after administration to a subject. In some embodiments, —X-L-R¹ iscleavable. In some embodiments, —X-L-R¹ is —S-L-R¹, and is converted to—S⁻ after administration to a subject. In some embodiments, theconversion is promoted by an enzyme of a subject. As appreciated by aperson skilled in the art, methods of determining whether the —S-L-R¹group is converted to —S⁻ after administration is widely known andpracticed in the art, including those used for studying drug metabolismand pharmacokinetics.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-a:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-b:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I is anphosphorothioate triester linkage having the structure of formula I-c:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,    —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl;-   each

-    independently represents a connection to a nucleoside; and-   R¹ is not —H when L is a covalent bond.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage having the structureof formula I-c is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more phosphate diesterlinkages, and one or more modified internucleotide linkages having theformula of I-a, I-b, or I-c.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage having the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least two phosphorothioate triester linkages having the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages having the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages having the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least five phosphorothioate triester linkages having the structure offormula I-c.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising asequence found in GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the saidsequence has over 50% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO:41). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the said sequence has over60% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 41), wherein the said sequence has over 70% identity withGGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising asequence found in GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the saidsequence has over 80% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO:41). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the said sequence has over90% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 41), wherein the said sequence has over 95% identity withGGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41). In some embodiments,the present disclosure provides a chirally controlled oligonucleotidehaving the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41).

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 41), wherein at least one internucleotidic linkage has thestructure of formula I. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide comprising a sequencefound in GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 41), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide comprising a sequencefound in GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 41), wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage is

In some embodiments the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising the sequence of GGCACAAGGGCACAGACTTC (SEQ IDNO: 41), wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage has the structure of formula I. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising the sequence of GGCACAAGGGCACAGACTTC (SEQ IDNO: 41), wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein at least one internucleotidic linkage has achiral linkage phosphorus. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least oneinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:41), wherein each internucleotidic linkage has the structure of formulaI. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each internucleotidiclinkage has the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at leastone internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein at least one linkage phosphorus is Rp. It isunderstood by a person of ordinary skill in the art that in certainembodiments wherein the chirally controlled oligonucleotide comprises anRNA sequence, each T is independently and optionally replaced with U. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:41), wherein each linkage phosphorus is Rp. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at leastone linkage phosphorus is Sp. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein each linkagephosphorus is Sp. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is ablockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is astereoblockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is aP-modification blockmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is alinkage blockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is analtmer. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein the oligonucleotide is a stereoaltmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:41), wherein the oligonucleotide is a P-modification altmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:41), wherein the oligonucleotide is a linkage altmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:41), wherein the oligonucleotide is a unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein theoligonucleotide is a stereounimer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein theoligonucleotide is a P-modification unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein theoligonucleotide is a linkage unimer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein theoligonucleotide is a gapmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein the oligonucleotide is askipmer.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 41), wherein at least onecytosine is optionally and independently replaced by 5-methylcytosine.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 41), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that one or more nucleotides comprise a phosphorus modificationprone to “autorelease” under certain conditions. That is, under certainconditions, a particular phosphorus modification is designed such thatit self-cleaves from the oligonucleotide to provide, e.g., a phosphatediester such as those found in naturally occurring DNA and RNA. In someembodiments, such a phosphorus modification has a structure of —O-L-R¹,wherein each of L and R¹ is independently as defined above and describedherein. In some embodiments, an autorelease group comprises a morpholinogroup. In some embodiments, an autorelease group is characterized by theability to deliver an agent to the internucleotidic phosphorus linker,which agent facilitates further modification of the phosphorus atom suchas, e.g., desulfurization. In some embodiments, the agent is water andthe further modification is hydrolysis to form a phosphate diester as isfound in naturally occurring DNA and RNA.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein(including, as non-limiting examples, any sequence disclosed in anyTable). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence having over 50%identity with any sequence disclosed herein. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 60% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising a sequence having over70% identity with any sequence disclosed herein. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 80% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising a sequence having over90% identity with any sequence disclosed herein. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 95% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage has a chiral linkagephosphorus. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage has a chiral linkagephosphorus. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage has a chiral linkage phosphorus.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage has the structure of formula I. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone internucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one linkage phosphorus is Rp. It is understood by a person ofordinary skill in the art that in certain embodiments wherein thechirally controlled oligonucleotide comprises an RNA sequence, each T isindependently and optionally replaced with U. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingany sequence disclosed herein, wherein each linkage phosphorus is Rp. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone linkage phosphorus is Sp. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein each linkage phosphorus is Sp. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a blockmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is astereoblockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having any sequence disclosedherein, wherein the oligonucleotide is a P-modification blockmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a linkage blockmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is an altmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a stereoaltmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is aP-modification altmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having any sequencedisclosed herein, wherein the oligonucleotide is a linkage altmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a unimer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having any sequencedisclosed herein, wherein the oligonucleotide is a stereounimer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a P-modification unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingany sequence disclosed herein, wherein the oligonucleotide is a linkageunimer. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinthe oligonucleotide is a gapmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is a skipmer.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach cytosine is optionally and independently replaced by5-methylcytosine. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having any sequence disclosedherein, wherein at least one cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein each cytosine is optionally andindependently replaced by 5-methylcytosine.

In various embodiments, any sequence disclosed herein can be combinedwith one or more of the following as disclosed herein or known in theart: pattern of backbone linkages; pattern of backbone chiral centers;and pattern of backbone P-modifications; pattern of base modification;pattern of sugar modification; pattern of backbone linkages; pattern ofbackbone chiral centers; and pattern of backbone P-modifications.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that the resulting pharmaceutical properties are improved throughone or more particular modifications at phosphorus. It is welldocumented in the art that certain oligonucleotides are rapidly degradedby nucleases and exhibit poor cellular uptake through the cytoplasmiccell membrane [Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13(28);3441-65; Wagner et al., Med. Res. Rev. (2000), 20(6):417-51; Peyrotteset al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin et al.,(1996), 43(1):196-208; Bologna et al., (2002), Antisense & Nucleic AcidDrug Development 12:33-41]. For instance, Vives et al., Nucleic AcidsResearch (1999), 27(20):4071-76, found that tert-butyl SATEpro-oligonucleotides displayed markedly increased cellular penetrationcompared to the parent oligonucleotide.

In some embodiments, a modification at a linkage phosphorus ischaracterized by its ability to be transformed to a phosphate diester,such as those present in naturally occurring DNA and RNA, by one or moreesterases, nucleases, and/or cytochrome P450 enzymes, including but notlimited to, those listed in Table 1A, below.

TABLE 1A Example enzymes. Family Gene CYP1 CYP1A1, CYP1A2, CYP1B1 CYP2CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19,CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 CYP3CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4 CYP4A11, CYP4A22, CYP4B1, CYP4F2,CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1 CYP5CYP5A1 CYP7 CYP7A1, CYP7B1 CYP8 CYP8A1 (prostacyclin synthase), CYP8B1(bile acid biosynthesis) CYP11 CYP11A1, CYP11B1, CYP11B2 CYP17 CYP17A1CYP19 CYP19A1 CYP20 CYP20A1 CYP21 CYP21A2 CYP24 CYP24A1 CYP26 CYP26A1,CYP26B1, CYP26C1 CYP27 CYP27A1 (bile acid biosynthesis), CYP27B1(vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknownfunction) CYP39 CYP39A1 CYP46 CYP46A1 CYP51 CYP51A1 (lanosterol 14-alphademethylase)

In some embodiments, a modification at phosphorus results in aP-modification moiety characterized in that it acts as a pro-drug, e.g.,the P-modification moiety facilitates delivery of an oligonucleotide toa desired location prior to removal. For instance, in some embodiments,a P-modification moiety results from PEGylation at the linkagephosphorus. One of skill in the relevant arts will appreciate thatvarious PEG chain lengths are useful and that the selection of chainlength will be determined in part by the result that is sought to beachieved by PEGylation. For instance, in some embodiments, PEGylation iseffected in order to reduce RES uptake and extend in vivo circulationlifetime of an oligonucleotide.

In some embodiments, a PEGylation reagent for use in accordance with thepresent disclosure is of a molecular weight of about 300 g/mol to about100,000 g/mol. In some embodiments, a PEGylation reagent is of amolecular weight of about 300 g/mol to about 10,000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 300g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent isof a molecular weight of about 500 g/mol. In some embodiments, aPEGylation reagent of a molecular weight of about 1000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 3000g/mol. In some embodiments, a PEGylation reagent is of a molecularweight of about 5000 g/mol.

In certain embodiments, a PEGylation reagent is PEG500. In certainembodiments, a PEGylation reagent is PEG1000. In certain embodiments, aPEGylation reagent is PEG3000. In certain embodiments, a PEGylationreagent is PEG5000.

In some embodiments, a P-modification moiety is characterized in that itacts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

In some embodiments, a P-modification moiety is characterized in that itacts as an agent which promotes cell entry and/or endosomal escape, suchas a membrane-disruptive lipid or peptide.

In some embodiments, a P-modification moiety is characterized in that itacts as a targeting agent. In some embodiments, a P-modification moietyis or comprises a targeting agent. The phrase “targeting agent,” as usedherein, is an entity that is associates with a payload of interest(e.g., with an oligonucleotide or oligonucleotide composition) and alsointeracts with a target site of interest so that the payload of interestis targeted to the target site of interest when associated with thetargeting agent to a materially greater extent than is observed underotherwise comparable conditions when the payload of interest is notassociated with the targeting agent. A targeting agent may be, orcomprise, any of a variety of chemical moieties, including, for example,small molecule moieties, nucleic acids, polypeptides, carbohydrates,etc. Targeting agents are described further by Adarsh et al., “OrganelleSpecific Targeted Drug Delivery—A Review,” International Journal ofResearch in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

Examples of such targeting agents include, but are not limited to,proteins (e.g. Transferrin), oligopeptides (e.g., cyclic and acrylicRGD-containing oligopeptides), antibodies (monoclonal and polyclonalantibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies),sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides(mannose, mannose-6-phosphate, galactose, and the like)), vitamins(e.g., folate), or other small biomolecules. In some embodiments, atargeting moiety is a steroid molecule (e.g., bile acids includingcholic acid, deoxycholic acid, dehydrocholic acid; cortisone;digoxigenin; testosterone; cholesterol; cationic steroids such ascortisone having a trimethylaminomethyl hydrazide group attached via adouble bond at the 3-position of the cortisone ring, etc.). In someembodiments, a targeting moiety is a lipophilic molecule (e.g.,alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes,terpenes, and polyalicyclic hydrocarbons such as adamantine andbuckminsterfullerenes). In some embodiments, a lipophilic molecule is aterpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal.In some embodiments, a targeting moiety is a peptide.

In some embodiments, a P-modification moiety is a targeting agent offormula —X-L-R¹ wherein each of X, L, and R¹ are as defined in Formula Iabove.

In some embodiments, a P-modification moiety is characterized in that itfacilitates cell specific delivery.

In some embodiments, a P-modification moiety is characterized in that itfalls into one or more of the above-described categories. For instance,in some embodiments, a P-modification moiety acts as a PK enhancer and atargeting ligand. In some embodiments, a P-modification moiety acts as apro-drug and an endosomal escape agent. One of skill in the relevantarts would recognize that numerous other such combinations are possibleand are contemplated by the present disclosure.

Nucleobases

In some embodiments, a nucleobase present in a provided oligonucleotideis a natural nucleobase or a modified nucleobase derived from a naturalnucleobase. Examples include, but are not limited to, uracil, thymine,adenine, cytosine, and guanine having their respective amino groupsprotected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine,5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidineanalogs such as pseudoisocytosine and pseudouracil and other modifiednucleobases such as 8-substituted purines, xanthine, or hypoxanthine(the latter two being the natural degradation products). Examplemodified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9,1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7,313.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

wherein R⁸ is an optionally substituted, linear or branched groupselected from aliphatic, aryl, aralkyl, aryloxylalkyl, carbocyclyl,heterocyclyl or heteroaryl group having 1 to 15 carbon atoms, including,by way of example only, a methyl, isopropyl, phenyl, benzyl, orphenoxymethyl group; and each of R⁹ and R¹⁰ is independently anoptionally substituted group selected from linear or branched aliphatic,carbocyclyl, aryl, heterocyclyl and heteroaryl.

Modified nucleobases also include expanded-size nucleobases in which oneor more aryl rings, such as phenyl rings, have been added. Nucleic basereplacements described in the Glen Research catalog(www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007, 40,141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., etal., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr.Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol.,2006, 10, 622-627, are contemplated as useful for the synthesis of thenucleic acids described herein. Some examples of these expanded-sizenucleobases are shown below:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett., 2002, 4, 4377-4380. Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

In some embodiments, modified nucleobases are of any one of thefollowing structures, optionally substituted:

In some embodiments, a modified nucleobase is fluorescent. Examples ofsuch fluorescent modified nucleobases include phenanthrene, pyrene,stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene,benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil,and naphtho-uracil, as shown below:

In some embodiments, a modified nucleobase is unsubstituted. In someembodiments, a modified nucleobase is substituted. In some embodiments,a modified nucleobase is substituted such that it contains, e.g.,heteroatoms, alkyl groups, or linking moieties connected to fluorescentmoieties, biotin or avidin moieties, or other protein or peptides. Insome embodiments, a modified nucleobase is a “universal base” that isnot a nucleobase in the most classical sense, but that functionssimilarly to a nucleobase. One representative example of such auniversal base is 3-nitropyrrole.

In some embodiments, other nucleosides can also be used in the processdisclosed herein and include nucleosides that incorporate modifiednucleobases, or nucleobases covalently bound to modified sugars. Someexamples of nucleosides that incorporate modified nucleobases include4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; dihydrouridine;2′-O-methylpseudouridine; beta,D-galactosylqueosine;2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine;1-methylpseudouridine; 1-methylguanosine; 1-methylinosine;2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine;N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine;5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine;N⁶-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine;5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine;5-methoxycarbonylmethyluridine; 5-methoxyuridine;2-methylthio-N⁶-isopentenyladenosine;N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;2-thiouridine; 4-thiouridine; 5-methyluridine;2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, nucleosides include 6′-modified bicyclic nucleosideanalogs that have either (R) or (S)-chirality at the 6′-position andinclude the analogs described in U.S. Pat. No. 7,399,845. In otherembodiments, nucleosides include 5′-modified bicyclic nucleoside analogsthat have either (R) or (S)-chirality at the 5′-position and include theanalogs described in US Patent Application Publication No. 20070287831.

In some embodiments, a nucleobase or modified nucleobase comprises oneor more biomolecule binding moieties such as e.g., antibodies, antibodyfragments, biotin, avidin, streptavidin, receptor ligands, or chelatingmoieties. In other embodiments, a nucleobase or modified nucleobase is5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments,a nucleobase or modified nucleobase is modified by substitution with afluorescent or biomolecule binding moiety. In some embodiments, thesubstituent on a nucleobase or modified nucleobase is a fluorescentmoiety. In some embodiments, the substituent on a nucleobase or modifiednucleobase is biotin or avidin.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is hereinincorporated by reference in its entirety.

In some embodiments, a base is optionally substituted A, T, C, G or U,wherein one or more —NH₂ are independently and optionally replaced with—C(-L-R¹)₃, one or more —NH— are independently and optionally replacedwith —C(-L-R¹)₂—, one or more ═N— are independently and optionallyreplaced with —C(-L-R¹)—, one or more ═CH— are independently andoptionally replaced with ═N—, and one or more ═O are independently andoptionally replaced with ═S, ═N(-L-R¹), or ═C(-L-R¹)₂, wherein two ormore -L-R¹ are optionally taken together with their intervening atoms toform a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatomring atoms. In some embodiments, a modified base is optionallysubstituted A, T, C, G or U, wherein one or more —NH₂ are independentlyand optionally replaced with —C(-L-R¹)₃, one or more —NH— areindependently and optionally replaced with —C(-L-R¹)₂—, one or more ═N—are independently and optionally replaced with —C(-L-R¹)—, one or more═CH— are independently and optionally replaced with ═N—, and one or more═O are independently and optionally replaced with ═S, ═N(-L-R¹), or═C(-L-R¹)₂, wherein two or more -L-R¹ are optionally taken together withtheir intervening atoms to form a 3-30 membered bicyclic or polycyclicring having 0-10 heteroatom ring atoms, wherein the modified base isdifferent than the natural A, T, C, G and U. In some embodiments, a baseis optionally substituted A, T, C, G or U. In some embodiments, amodified base is substituted A, T, C, G or U, wherein the modified baseis different than the natural A, T, C, G and U.

In some embodiments, a modified nucleotide or nucleotide analog is anymodified nucleotide or nucleotide analog described in any of: Gryaznov,S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsenet al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58,2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al.1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let.8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen etal. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew.Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res.Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76;Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1:3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika etal. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm.48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshiet al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic AcidsRes. 24: 2966; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al.2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75:1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth etal. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew;Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P;Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; etal. From Nucleic Acids Symposium Series (2008), 52(1), 553-554; Singh etal. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63:10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003Chem. Comm. 2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220;Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseuret al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO20070900071; or WO 2016/079181.

Sugars

In some embodiments, provided oligonucleotides comprise one or moremodified sugar moieties beside the natural sugar moieties.

The most common naturally occurring nucleotides are comprised of ribosesugars linked to the nucleobases adenosine (A), cytosine (C), guanine(G), and thymine (T) or uracil (U). Also contemplated are modifiednucleotides wherein a phosphate group or linkage phosphorus in thenucleotides can be linked to various positions of a sugar or modifiedsugar. As non-limiting examples, the phosphate group or linkagephosphorus can be linked to the 2′, 3′, 4′ or 5′ hydroxyl moiety of asugar or modified sugar. Nucleotides that incorporate modifiednucleobases as described herein are also contemplated in this context.In some embodiments, nucleotides or modified nucleotides comprising anunprotected —OH moiety are used in accordance with methods of thepresent disclosure.

Other modified sugars can also be incorporated within a providedoligonucleotide. In some embodiments, a modified sugar contains one ormore substituents at the 2′ position including one of the following: —F;—CF₃, —CN, —N₃, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ isindependently as defined above and described herein; —O—(C₁-C₁₀ alkyl),—S—(C₁-C₁₀ alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀alkenyl), —S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀alkenyl)₂; —O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀alkylene)-NH(C₁-C₁₀ alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or—N(C₁-C₁₀ alkyl)-(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl,alkylene, alkenyl and alkynyl may be substituted or unsubstituted.Examples of substituents include, and are not limited to,—O(CH₂)_(n)OCH₃, and —O(CH₂)_(n)NH₂, wherein n is from 1 to about 10,MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugarsdescribed in WO 2001/088198; and Martin et al., Helv. Chim. Acta, 1995,78, 486-504. In some embodiments, a modified sugar comprises one or moregroups selected from a substituted silyl group, an RNA cleaving group, areporter group, a fluorescent label, an intercalator, a group forimproving the pharmacokinetic properties of a nucleic acid, a group forimproving the pharmacodynamic properties of a nucleic acid, or othersubstituents having similar properties. In some embodiments,modifications are made at one or more of the the 2′, 3′, 4′, 5′, or 6′positions of the sugar or modified sugar, including the 3′ position ofthe sugar on the 3′-terminal nucleotide or in the 5′ position of the5′-terminal nucleotide.

In some embodiments, the 2′-OH of a ribose is replaced with asubstituent including one of the following: —H, —F; —CF₃, —CN, —N₃, —NO,—NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently asdefined above and described herein; —O—(C₁-C₁₀ alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀ alkenyl),—S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀ alkenyl)₂;—O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀ alkynyl), or—N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl),—O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀ alkylene)-NH(C₁-C₁₀alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀alkyl)-(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl, alkylene,alkenyl and alkynyl may be substituted or unsubstituted. In someembodiments, the 2′-OH is replaced with —H (deoxyribose). In someembodiments, the 2′-OH is replaced with —F. In some embodiments, the2′-OH is replaced with —OR′. In some embodiments, the 2′-OH is replacedwith —OMe. In some embodiments, the 2′-OH is replaced with —OCH₂CH₂OMe.

Modified sugars also include locked nucleic acids (LNAs). In someembodiments, two substituents on sugar carbon atoms are taken togetherto form a bivalent moiety. In some embodiments, two substituents are ontwo different sugar carbon atoms. In some embodiments, a formed bivalentmoiety has the structure of -L- as defined herein. In some embodiments,-L- is —O—CH₂—, wherein —CH₂— is optionally substituted. In someembodiments, -L- is —O—CH₂—. In some embodiments, -L- is —O—CH(Et)-. Insome embodiments, -L- is between C2 and C4 of a sugar moiety. In someembodiments, a locked nucleic acid has the structure indicated below. Alocked nucleic acid of the structure below is indicated, wherein Barepresents a nucleobase or modified nucleobase as described herein, andwherein R^(2s) is —OCH₂C4′-.

In some embodiments, a modified sugar is an ENA such as those describedin, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132(42):14942-14950. In some embodiments, a modified sugar is any of those foundin an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol,threose, 2′fluoroarabinose, or cyclohexene.

Modified sugars include sugar mimetics such as cyclobutyl or cyclopentylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; and 5,359,044. Some modified sugars that arecontemplated include sugars in which the oxygen atom within the ribosering is replaced by nitrogen, sulfur, selenium, or carbon. In someembodiments, a modified sugar is a modified ribose wherein the oxygenatom within the ribose ring is replaced with nitrogen, and wherein thenitrogen is optionally substituted with an alkyl group (e.g., methyl,ethyl, isopropyl, etc).

Non-limiting examples of modified sugars include glycerol, which formglycerol nucleic acid (GNA) analogues. One example of a GNA analogue isshown below and is described in Zhang, R et al., J. Am. Chem. Soc.,2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127,4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603 (X═O⁻):

Another example of a GNA derived analogue, flexible nucleic acid (FNA)based on the mixed acetal aminal of formyl glycerol, is described inJoyce G F et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D andSwitzer C, J. Am. Chem. Soc., 2008, 130, 412-413, and is shown below:

Additional non-limiting examples of modified sugars includehexopyranosyl (6′ to 4′), pentopyranosyl (4′ to 2′), pentopyranosyl (4′to 3′), or tetrofuranosyl (3′ to 2′) sugars. In some embodiments, ahexopyranosyl (6′ to 4′) sugar is of any one in the following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 2′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 3′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a tetrofuranosyl (3′ to 2′) sugar is of either inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a modified sugar is of any one in the followingformulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, one or more hydroxyl group in a sugar moiety isoptionally and independently replaced with halogen, R′—N(R′)₂, —OR′, or—SR′, wherein each R′ is independently as defined above and describedherein.

In some embodiments, a sugar mimetic is as illustrated below, whereinX^(s) corresponds to the P-modification group “—XLR¹” described herein,Ba is as defined herein, and X¹ is selected from —S—, —Se—, —CH₂—,—NMe-, —NEt- or —NiPr—.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43% 44%, 45%, 46%, 47%, 48%, 49%, 50% or more (e.g.,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more), inclusive, of thesugars in a chirally controlled oligonucleotide composition aremodified. In some embodiments, only purine residues are modified (e.g.,about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more] of the purine residues are modified).In some embodiments, only pyrimidine residues are modified (e.g., about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more] of the pyridimine residues are modified). Insome embodiments, both purine and pyrimidine residues are modified.

Modified sugars and sugar mimetics can be prepared by methods known inthe art, including, but not limited to: A. Eschenmoser, Science (1999),284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M.Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoserin Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V.Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U.Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al,Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta(1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K.Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser,Science (1999), 284:2118. Modifications to the 2′ modifications can befound in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and allreferences therein. Specific modifications to the ribose can be found inthe following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem.,1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79,1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310). Insome embodiments, a modified sugar is any of those described in PCTPublication No. WO2012/030683, incorporated herein by reference, anddepicted in the FIGS. 26-30 of the present application. In someembodiments, a modified sugar is any modified sugar described in any of:Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix etal. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4:5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem.1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273;Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo.Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5:530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256;Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Morita etal. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo.Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett.2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett.39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen etal. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun.1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al.2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53:8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al.2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc.Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R;Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth;Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From NucleicAcids Symposium Series (2008), 52(1), 553-554; Singh et al. 1998 Chem.Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh etal. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm.2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; VanAerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur etal. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071;or WO 2016/079181.

In some embodiments, a modified sugar moiety is an optionallysubstituted pentose or hexose moiety. In some embodiments, a modifiedsugar moiety is an optionally substituted pentose moiety. In someembodiments, a modified sugar moiety is an optionally substituted hexosemoiety. In some embodiments, a modified sugar moiety is an optionallysubstituted ribose or hexitol moiety. In some embodiments, a modifiedsugar moiety is an optionally substituted ribose moiety. In someembodiments, a modified sugar moiety is an optionally substitutedhexitol moiety.

In some embodiments, an example modified internucleotidic linkage and/orsugar is selected from:

In some embodiments, R¹ is R as defined and described. In someembodiments, R² is R. In some embodiments, R^(e) is R. In someembodiments, R^(e) is H, CH₃, Bn, COCF₃, benzoyl, benzyl,pyren-1-ylcarbonyl, pyren-1-ylmethyl, 2-aminoethyl. In some embodiments,an example modified internucleotidic linkage and/or sugar is selectedfrom those described in Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507,220; Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143;Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Jones etal. J. Org. Chem. 1993, 58, 2983; Vasseur et al. J. Am. Chem. Soc. 1992,114, 4006; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34:1338; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Koshkin et al. 1998Tetrahedron 54: 3607-3630; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5;Nielsen et al. 1997 Chem. Soc. Rev. 73; Schultz et al. 1996 NucleicAcids Res. 24: 2966; Obika et al. 1997 Tetrahedron Lett. 38 (50):8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Singh et al.1998 Chem. Comm. 1247-1248; Kumar et al. 1998 Bioo. Med. Chem. Let. 8:2219-2222; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1:3423-3433; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Seth et al.2010 J. Org. Chem. 75: 1569-1581; Singh et al. 1998 J. Org. Chem. 63:10035-39; Sorensen 2003 Chem. Comm. 2130-2131; Petersen et al. 2003TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun.1395-1396; Jepsen et al. 2004 Oligo. 14: 130-146; Morita et al. 2001Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem.Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226;Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Lauritsen et al. 2002Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13:253-256; WO 20070900071; Seth et al., Nucleic Acids Symposium Series(2008), 52(1), 553-554; Seth et al. 2009 J. Med. Chem. 52: 10-13; Sethet al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Pallan et al. 2012 Chem. Comm.48: 8195-8197; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al.2012 Bioo. Med. Chem. Lett. 22: 296-299; WO 2016/079181; U.S. Pat. Nos.6,326,199; 6,066,500; and 6,440,739, the base and sugar modifications ofeach of which is herein incorporated by reference.

Oligonucleotides

In some embodiments, the present disclosure provides oligonucleotidesand oligonucleotide compositions that are chirally controlled. Forinstance, in some embodiments, a provided composition containspredetermined levels of one or more individual oligonucleotide types,wherein an oligonucleotide type is defined by: 1) base sequence; 2)pattern of backbone linkages; 3) pattern of backbone chiral centers; and4) pattern of backbone P-modifications. In some embodiments, aparticular oligonucleotide type may be defined by 1A) base identity; 1B)pattern of base modification; 1C) pattern of sugar modification; 2)pattern of backbone linkages; 3) pattern of backbone chiral centers; and4) pattern of backbone P-modifications. In some embodiments,oligonucleotides of the same oligonucleotide type are identical.

As described herein, the present disclosure provides variousoligonucleotides. In some embodiments, the present disclosure providesoligonucleotides comprising a sequence that shares greater than about50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% identity with a sequence found ina provided example oligonucleotide, such as those listed in varioustables. In some embodiments, a provided oligonucleotide is WV-1092. Insome embodiments, a provided oligonucleotide is WV-2595. In someembodiments, a provided oligonucleotide is WV-2603. In some embodiments,the present disclosure provides oligonucleotides comprising orconsisting of a sequence found in a provided example oligonucleotide. Insome embodiments, the present disclosure provides oligonucleotidescomprising or consisting of a sequence found in WV-1092. In someembodiments, the present disclosure provides oligonucleotides comprisingor consisting of a sequence found in WV-2595. In some embodiments, thepresent disclosure provides oligonucleotides comprising or consisting ofa sequence found in WV-2603. In some embodiments, a providedoligonucleotide further comprises one or more natural phosphate linkagesand one or more modified internucleotidic linkages. In some embodiments,a provided oligonucleotide comprises two or more natural phosphatelinkages. In some embodiments, a provided oligonucleotide comprises twoor more consecutive natural phosphate linkages. In some embodiments, aprovided oligonucleotide comprises two or more modified internucleotidiclinkages. In some embodiments, a provided oligonucleotide comprises twoor more consecutive modified internucleotidic linkages. In someembodiments, a provided oligonucleotide comprises 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 5 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 5 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 6 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 7 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 8 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 9 or more consecutive modifiedinternucleotidic linkages. In some embodiments, a providedoligonucleotide comprises 10 or more consecutive modifiedinternucleotidic linkages. In some embodiments, at least one of themodified internucleotidic linkages is a chirally controlledinternucleotidic linkage in that oligonucleotides having the samesequence and chemical modifications within a composition share the sameconfiguration, either Rp or Sp, at the chiral phosphorus atom of themodified internucleotidic linkage. In some embodiments, at least twomodified internucleotidic linkages are chirally controlled. In someembodiments, at least one modified internucleotidic linkage within aconsecutive modified internucleotidic linkage region is chirallycontrolled. In some embodiments, at least two modified internucleotidiclinkages within a consecutive modified internucleotidic linkage regionare chirally controlled. In some embodiments, each modifiedinternucleotidic linkage within a consecutive modified internucleotidiclinkage region is chirally controlled. In some embodiments, eachmodified internucleotidic linkage is chirally controlled. In someembodiments, a provided oligonucleotide comprises a (Sp)xRp(Sp)ypattern, wherein each of x and y is independently 1-20, and the sum of xand y is 1-50. In some embodiments, each of x and y is independently2-20. In some embodiments, at least one of x and y is greater than 5, 6,7, 8, 9, or 10. In some embodiments, the sum of x and y is greater than5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In someembodiments, a provided oligonucleotide comprises one or more chemicalmodifications as presented in a provided example oligonucleotide. Insome embodiments, a provided oligonucleotide comprises one or more basemodifications as presented in a provided example oligonucleotide. Insome embodiments, a provided oligonucleotide comprises one or more sugarmodifications as presented in a provided example oligonucleotide. Insome embodiments, a sugar modification is a 2′-modification. In someembodiments, a sugar modification is LNA. In some embodiments, a sugarmodification is ENA. In some embodiments, a provided oligonucleotide isa chirally controlled oligonucleotide. In some embodiments, the presentdisclosure provides an oligonucleotide composition comprising a providedoligonucleotide. In some embodiments, a provided oligonucleotidecomposition is a chirally controlled oligonucleotide composition.

In some embodiments, a provided oligonucleotide is a unimer. In someembodiments, a provided oligonucleotide is a P-modification unimer. Insome embodiments, a provided oligonucleotide is a stereounimer. In someembodiments, a provided oligonucleotide is a stereounimer ofconfiguration Rp. In some embodiments, a provided oligonucleotide is astereounimer of configuration Sp.

In some embodiments, a provided oligonucleotide is an altmer. In someembodiments, a provided oligonucleotide is a P-modification altmer. Insome embodiments, a provided oligonucleotide is a stereoaltmer.

In some embodiments, a provided oligonucleotide is a blockmer. In someembodiments, a provided oligonucleotide is a P-modification blockmer. Insome embodiments, a provided oligonucleotide is a stereoblockmer.

In some embodiments, a provided oligonucleotide is a gapmer.

In some embodiments, a provided oligonucleotide is a skipmer.

In some embodiments, a provided oligonucleotide is a hemimer. In someembodiments, a hemimer is an oligonucleotide wherein the 5′-end or the3′-end has a sequence that possesses a structure feature that the restof the oligonucleotide does not have. In some embodiments, the 5′-end orthe 3′-end has or comprises 2 to 20 nucleotides. In some embodiments, astructural feature is a base modification. In some embodiments, astructural feature is a sugar modification. In some embodiments, astructural feature is a P-modification. In some embodiments, astructural feature is stereochemistry of the chiral internucleotidiclinkage. In some embodiments, a structural feature is or comprises abase modification, a sugar modification, a P-modification, orstereochemistry of the chiral internucleotidic linkage, or combinationsthereof. In some embodiments, a hemimer is an oligonucleotide in whicheach sugar moiety of the 5′-end sequence shares a common modification.In some embodiments, a hemimer is an oligonucleotide in which each sugarmoiety of the 3′-end sequence shares a common modification. In someembodiments, a common sugar modification of the 5′ or 3′ end sequence isnot shared by any other sugar moieties in the oligonucleotide. In someembodiments, an example hemimer is an oligonucleotide comprising asequence of substituted or unsubstituted 2′-O-alkyl sugar modifiednucleosides, bicyclic sugar modified nucleosides, β-D-ribonucleosides orβ-D-deoxyribonucleosides (for example 2′-MOE modified nucleosides, andLNA™ or ENA™ bicyclic sugar modified nucleosides) at one terminus and asequence of nucleosides with a different sugar moiety (such as asubstituted or unsubstituted 2′-O-alkyl sugar modified nucleosides,bicyclic sugar modified nucleosides or natural ones) at the otherterminus. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, hemimerand skipmer. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, andskipmer. For instance, in some embodiments, a provided oligonucleotideis both an altmer and a gapmer. In some embodiments, a providednucleotide is both a gapmer and a skipmer. One of skill in the chemicaland synthetic arts will recognize that numerous other combinations ofpatterns are available and are limited only by the commercialavailability and/or synthetic accessibility of constituent partsrequired to synthesize a provided oligonucleotide in accordance withmethods of the present disclosure. In some embodiments, a hemimerstructure provides advantageous benefits, as exemplified by FIG. 29. Insome embodiments, provided oligonucleotides are 5′-hemmimers thatcomprises modified sugar moieties in a 5′-end sequence. In someembodiments, provided oligonucleotides are 5′-hemmimers that comprisesmodified 2′-sugar moieties in a 5′-end sequence.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleotides. In some embodiments, a providedoligonucleotide comprises one or more modified nucleotides. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted nucleosides. In some embodiments, a provided oligonucleotidecomprises one or more modified nucleosides. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedLNAs.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleobases. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted naturalnucleobases. In some embodiments, a provided oligonucleotide comprisesone or more optionally substituted modified nucleobases. In someembodiments, a provided oligonucleotide comprises one or more5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or5-carboxylcytosine. In some embodiments, a provided oligonucleotidecomprises one or more 5-methylcytidine.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted sugars. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted sugarsfound in naturally occurring DNA and RNA. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedribose or deoxyribose. In some embodiments, a provided oligonucleotidecomprises one or more optionally substituted ribose or deoxyribose,wherein one or more hydroxyl groups of the ribose or deoxyribose moietyis optionally and independently replaced by halogen, R′, —N(R′)₂, —OR′,or —SR′, wherein each R′ is independently as defined above and describedherein. In some embodiments, a provided oligonucleotide comprises one ormore optionally substituted deoxyribose, wherein the 2′ position of thedeoxyribose is optionally and independently substituted with halogen,R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as definedabove and described herein. In some embodiments, a providedoligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with halogen. In some embodiments, aprovided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with one or more —F. halogen. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently as defined above and described herein. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ aliphatic. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ alkyl. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OMe. In some embodiments,a provided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with —O-methoxyethyl.

In some embodiments, a provided oligonucleotide is single-strandedoligonucleotide.

In some embodiments, a provided oligonucleotide is a hybridizedoligonucleotide strand. In certain embodiments, a providedoligonucleotide is a partially hybridized oligonucleotide strand. Incertain embodiments, a provided oligonucleotide is a completelyhybridized oligonucleotide strand. In certain embodiments, a providedoligonucleotide is a double-stranded oligonucleotide. In certainembodiments, a provided oligonucleotide is a triple-strandedoligonucleotide (e.g., a triplex).

In some embodiments, a provided oligonucleotide is chimeric. Forexample, in some embodiments, a provided oligonucleotide is DNA-RNAchimera, DNA-LNA chimera, etc.

In some embodiments, any one of the structures comprising anoligonucleotide depicted in WO2012/030683 can be modified in accordancewith methods of the present disclosure to provide chirally controlledvariants thereof. For example, in some embodiments the chirallycontrolled variants comprise a stereochemical modification at any one ormore of the linkage phosphorus and/or a P-modification at any one ormore of the linkage phosphorus. For example, in some embodiments, aparticular nucleotide unit of an oligonucleotide of WO2012/030683 ispreselected to be stereochemically modified at the linkage phosphorus ofthat nucleotide unit and/or P-modified at the linkage phosphorus of thatnucleotide unit. In some embodiments, a chirally controlledoligonucleotide is of any one of the structures depicted in FIGS. 26-30.In some embodiments, a chirally controlled oligonucleotide is a variant(e.g., modified version) of any one of the structures depicted in FIGS.26-30. The disclosure of WO2012/030683 is herein incorporated byreference in its entirety.

In some embodiments, a provided oligonucleotide is a therapeutic agent.

In some embodiments, a provided oligonucleotide is an antisenseoligonucleotide.

In some embodiments, a provided oligonucleotide is an antigeneoligonucleotide.

In some embodiments, a provided oligonucleotide is a decoyoligonucleotide.

In some embodiments, a provided oligonucleotide is part of a DNAvaccine.

In some embodiments, a provided oligonucleotide is an immunomodulatoryoligonucleotide, e.g., immunostimulatory oligonucleotide andimmunoinhibitory oligonucleotide.

In some embodiments, a provided oligonucleotide is an adjuvant.

In some embodiments, a provided oligonucleotide is an aptamer.

In some embodiments, a provided oligonucleotide is a ribozyme.

In some embodiments, a provided oligonucleotide is a deoxyribozyme(DNAzymes or DNA enzymes).

In some embodiments, a provided oligonucleotide is an siRNA.

In some embodiments, a provided oligonucleotide is a microRNA, or miRNA.

In some embodiments, a provided oligonucleotide is a ncRNA (non-codingRNAs), including a long non-coding RNA (lncRNA) and a small non-codingRNA, such as piwi-interacting RNA (piRNA).

In some embodiments, a provided oligonucleotide is complementary to astructural RNA, e.g., tRNA.

In some embodiments, a provided oligonucleotide is a nucleic acidanalog, e.g., GNA, LNA, PNA, TNA, GNA, ANA, FANA, CeNA, HNA, UNA, ZNA,or Morpholino.

In some embodiments, a provided oligonucleotide is a P-modified prodrug.

In some embodiments, a provided oligonucleotide is a primer. In someembodiments, a primers is for use in polymerase-based chain reactions(i.e., PCR) to amplify nucleic acids. In some embodiments, a primer isfor use in any known variations of PCR, such as reverse transcriptionPCR (RT-PCR) and real-time PCR.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to modulate RNase H activation. For example, in someembodiments, RNase H activation is modulated by the presence ofstereocontrolled phosphorothioate nucleic acid analogs, with naturalDNA/RNA being more or equally susceptible than the Rp stereoisomer,which in turn is more susceptible than the corresponding Spstereoisomer.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to indirectly or directly increase or decreaseactivity of a protein or inhibition or promotion of the expression of aprotein. In some embodiments, a provided oligonucleotide ischaracterized in that it is useful in the control of cell proliferation,viral replication, and/or any other cell signaling process.

In some embodiments, a provided oligonucleotide is from about 2 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 4 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 5 to about10 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 10 to about 30 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 15 toabout 25 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

In some embodiments, an oligonucleotide is at least 2 nucleotide unitsin length. In some embodiments, an oligonucleotide is at least 3nucleotide units in length. In some embodiments, an oligonucleotide isat least 4 nucleotide units in length. In some embodiments, anoligonucleotide is at least 5 nucleotide units in length. In someembodiments, an oligonucleotide is at least 6 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 7 nucleotideunits in length. In some embodiments, an oligonucleotide is at least 8nucleotide units in length. In some embodiments, an oligonucleotide isat least 9 nucleotide units in length. In some embodiments, anoligonucleotide is at least 10 nucleotide units in length. In someembodiments, an oligonucleotide is at least 11 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 12nucleotide units in length. In some embodiments, an oligonucleotide isat least 13 nucleotide units in length. In some embodiments, anoligonucleotide is at least 14 nucleotide units in length. In someembodiments, an oligonucleotide is at least 15 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 16nucleotide units in length. In some embodiments, an oligonucleotide isat least 17 nucleotide units in length. In some embodiments, anoligonucleotide is at least 18 nucleotide units in length. In someembodiments, an oligonucleotide is at least 19 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 20nucleotide units in length. In some embodiments, an oligonucleotide isat least 21 nucleotide units in length. In some embodiments, anoligonucleotide is at least 22 nucleotide units in length. In someembodiments, an oligonucleotide is at least 23 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 24nucleotide units in length. In some embodiments, an oligonucleotide isat least 25 nucleotide units in length. In some other embodiments, anoligonucleotide is at least 30 nucleotide units in length. In some otherembodiments, an oligonucleotide is a duplex of complementary strands ofat least 18 nucleotide units in length. In some other embodiments, anoligonucleotide is a duplex of complementary strands of at least 21nucleotide units in length.

In some embodiments, the 5′-end and/or the 3′-end of a providedoligonucleotide is modified. In some embodiments, the 5′-end and/or the3′-end of a provided oligonucleotide is modified with a terminal capmoiety. Examples of such modifications, including terminal cap moietiesare extensively described herein and in the art, for example but notlimited to those described in US Patent Application Publication US2009/0023675A1.

In some embodiments, oligonucleotides of an oligonucleotide typecharacterized by 1) a common base sequence and length, 2) a commonpattern of backbone linkages, and 3) a common pattern of backbone chiralcenters, have the same chemical structure. For example, they have thesame base sequence, the same pattern of nucleoside modifications, thesame pattern of backbone linkages (i.e., pattern of internucleotidiclinkage types, for example, phosphate, phosphorothioate, etc), the samepattern of backbone chiral centers (i.e. pattern of linkage phosphorusstereochemistry (Rp/Sp)), and the same pattern of backbone phosphorusmodifications (e.g., pattern of “—XLR¹” groups in formula I). ExampleOligonucleotides and Compositions

In some embodiments, a provided chirally controlled oligonucleotidecomprises the sequence of, or part of the sequence of mipomersen.Mipomersen is based on the following base sequenceGCCT/UCAGT/UCT/UGCT/UT/UCGCACC (SEQ ID NO: 42). In some embodiments, oneor more of any of the nucleotide or linkages may be modified inaccordance of the present disclosure. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence ofG*-C*-C*-U*-C*-dA-dG-dT-dC-dT-dG-dmC-dT-dT-dmC-G*-C*-A*-C*-C* (SEQ IDNO: 43) [d=2′-deoxy, *=2′-O-(2-methoxyethyl)] with 3′→5′phosphorothioate linkages. Example modified mipomersen sequences aredescribed throughout the application, including but not limited to thosein Table 2.

In certain embodiments, a provided oligonucleotide is a mipomersenunimer. In certain embodiments, a provided oligonucleotide is amipomersen unimer of configuration Rp. In certain embodiments, aprovided oligonucleotide is a mipomersen unimer of configuration Sp.

Exemplary chirally controlled oligonucleotides comprising the sequenceof, or part of the sequence of mipomersen is depicted in Table 2, below.

TABLE 2 Example oligonucleotides. SEQ ID Oligo Stereochemistry/SequenceDescription NO: 101 All-(Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC]All-R 44 102 All-(Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] All-S45 103 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp, Rp, 5R-9S-5R 46 Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 104(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp,5S-9R-5S 47 Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 105 (Sp, Rp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp, 1S-17R-1S 48Rp, Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 106 (Rp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 1R-17S-1R 49Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 107 (Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, (R/S)₉R 50Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 108 (Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (S/R)₉S 51Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 109 (Sp, Sp, Sp, Rp, Rp,Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, 3S-13R-3S 52Sp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 110 (Rp, Rp, Rp, Sp, Sp,Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, 3R-13S-3R 53Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 111 (Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R¹⁹ 54Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 112 (Sp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Rp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R⁹ 55Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 113 (Sp, Rp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R² 56Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 114 (Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, (RRS)₆-R 57Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 115 (Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, S-(RRS)₆ 58Sp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] 116 (Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 59Rp)d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC] (RRS)₅-RR 122 All-(Rp)-All-R 60 d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1 As1Cs1C]123 (Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Rp, Rp, Rp,1S-17R-1S 61 Rp, Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 124All-(Sp)-d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1 All-S 62Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] 126 All-(Rp)-d[Cs2As2Gs2T] All-R 127All-(Rp)-d[Cs3As3Gs3T] All-R 128 All-(Sp)-d[Cs4As4Gs4T] All-S 129All-(Sp)-d[Cs5As5Gs5T] All-S 130 All-(Sp)-d[Cs6As6Gs6T] All-S 131All-(Rp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-R 63Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 132All-(Sp)-d[Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7 All-S 64Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] 133 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, SpSp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, 5R-9S-5R 65Rp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 134 (Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, 5S-9R-5S 66Sp)-d[Gs15mCs15mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mCs1As15mCs15mC] 135All-(Rp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-R 67 136All-(Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] All-S 68 137 (Sp,Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp)- 1S-9R-1S 69d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 138 (Sp, Sp, Rp, Rp, Rp,Rp, Rp, Rp, Rp, Sp, Sp)- 2S-7R-2S 70d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 139 (Rp, Sp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Sp, Rp)- 1R-9S-1R 71d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 140 (Rp, Rp, Sp, Sp, Sp,Sp, Sp, Sp, Sp, Rp, Rp)- 2R-7S-2R 72d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 141 (Sp, Sp, Sp, Rp, Rp,Rp, Rp, Rp, Sp, Sp, Sp)- 3S-5R-3S 73d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 142 (Rp, Rp, Rp, Sp, Sp,Sp, Sp, Sp, Rp, Rp, Rp)- 3R-5S-3R 74d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 143 (Sp, Sp, Rp, Sp, Sp,Rp, Sp, Sp, Rp, Sp, Sp)- (SSR)₃-SS 75d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 144 (Rp, Rp, Sp, Rp, Rp,Sp, Rp, Rp, Sp, Rp, Rp)- (RRS)₃-RR 76d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] 145 All-(Rp)- All-R 77d[5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1 Gs15mC] 146All-(Rp)-d[Gs15mCs1Ts1G] All-R 147 All-(Rp)-d[5mCs1As1Gs1T] All-R 148All-(Rp)-d[5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G] All-R 78 149All-(Rp)-d[5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G] All-R 79 151All-(Sp)-d[Cs1AsGs1T] All-S 152 All-(Sp)-d[Cs1AGs1T] All-S 153All-(Sp)-d[CAs1GsT] All-S 157 All-(Sp)-d[5mCs1As1Gs1T] All-S 158 (Sp,Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S 80 d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCs1GsCsACsC] 159 (Sp, Sp, Sp,Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, 5S-9R-5S 81Sp)-d[Gs1Cs1Cs1Ts1CsAsGsTsCsTsGsCsTsTsCs1GsCs2As2Cs2C] 160 All-(Rp)-All-R 82 (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 161 All-(Sp)- All-S 83(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 162 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp,Sp, Sp, Sp, Rp, Rp, Rp, Rp, 5R-9S-5R 84 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 163 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp, 5S-9R-5S 85Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 164 (Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Rp, Rp, Rp, 1S-17R-1S 86 Rp, Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 165 (Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, 1R-17S-1R 87 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 166 (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, (R/S)₉R 88 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 167 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, (S/R)₉S 89Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 168 (Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Rp, Rp, Sp, Sp, 3S-13R-3S 90Sp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 169 (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp,Sp, Sp, Sp, Sp, Sp, Rp, Rp, 3R-13S-3R 91 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 170 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R¹⁹ 92 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 171 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R⁹ 93Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 172 (Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp Sp,Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S/R² 94Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 173 (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, (RRS)₆-R 95 Rp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 174 (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, RpRp, Sp, Rp, Rp, Sp, Rp, Rp, S-(RRS)₆ 96Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) 175 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 97Rp)(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs] (RRS)₅-RR(Gs5mCsAs5mCs5mC)_(MOE) 176 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp,Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 98Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[As1Gs1Ts15mCs1Ts1Gs15m (RRS)₅-RRCs1Ts1Ts15mCs1] (Gs15mCs1As15mCs15mC)_(MOE) 177 (Rp, Sp, Rp, Rp, Sp, Rp,Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 99Rp)(Gs15mCs15mCs1Ts15mCs1)_(MOE)d[AGT5mCTG5mCTT5mC] (RRS)₅-RR(Gs25mCs2As25mCs25mC)_(MOE) 178 (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp,Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, S-(RRS)₆ 100Sp)-(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(F) (F: 2-fluorodeoxyribose) 179 (Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 101Rp)d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs (RRS)₅-RR8As8Cs8C] 180 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp RS- 102Rp)d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs (RRS)₅-RR9As9Cs9C] 181 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp, Sp, Rp, Rp, Sp,Rp, Rp, Sp, Rp RS- 103Rp)d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts (RRS)₅-RR10Ts10Cs10Gs10Cs10As10Cs10C] 182 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 104Rp)d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts (RRS)₅-RR11Ts11Cs11Gs11Cs11As11Cs11C] 183 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 105Rp)d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts (RRS)₅-RR12Ts12Cs12Gs12Cs12As12Cs12C] 184 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 106Rp)d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts (RRS)₅-RR13Ts13Cs13Gs13Cs13As13Cs13C] 185 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 107Rp)d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts (RRS)₅-RR14Ts14Cs14Gs14Cs14As14Cs14C] 186 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 108Rp)d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts (RRS)₅-RR15Ts15Cs15Gs15Cs15As15Cs15C] 187 (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp Rp,Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp RS- 109Rp)d[GsCsCs1TsCsAs]GsUs2CsUsGsd[CsTs3TsCsGs]CsAs4CsC (RRS)₅-RR 188 (Sp,Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S 110 d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsACsC] 189 (Sp, Sp, Sp,Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 111d[Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1CsAC s1C] 190 (Sp, Sp,Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S112 d[Gs8Cs8Cs8Ts8Cs8As8Gs8Ts8Cs8Ts8Gs8Cs8Ts8Ts8Cs8Gs8Cs1A Cs8C] 191(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S 113 d[Gs9Cs9Cs9Ts9Cs9As9Gs9Ts9Cs9Ts9Gs9Cs9Ts9Ts9Cs9Gs9Cs1ACs9C] 192 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp,Sp, Sp, Sp)- 5S-9R-4S 114d[Gs10Cs10Cs10Ts10Cs10As10Gs10Ts10Cs10Ts10Gs10Cs10Ts10Ts10Cs10Gs10Cs1ACs10C] 193 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 115d[Gs11Cs11Cs11Ts11Cs11As11Gs11Ts11Cs11Ts11Gs11Cs11Ts11Ts11Cs11Gs11Cs1ACs11C] 194 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 116d[Gs12Cs12Cs12Ts12Cs12As12Gs12Ts12Cs12Ts12Gs12Cs12Ts12Ts12Cs12Gs12Cs1ACs12C] 195 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 117d[Gs13Cs13Cs13Ts13Cs13As13Gs13Ts13Cs13Ts13Gs13Cs13Ts13Ts13Cs13Gs13Cs1ACs13C] 196 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 118d[Gs14Cs14Cs14Ts14Cs14As14Gs14Ts14Cs14Ts14Gs14Cs14Ts14Ts14Cs14Gs14Cs1ACs14C] 197 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 119d[Gs15Cs15Cs15Ts15Cs15As15Gs15Ts15Cs15Ts15Gs15Cs15Ts15Ts15Cs15Gs15Cs1ACs15C] 198 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp,Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 120GsCsCsUsCsAsGsUsCsUsGsCsUsUsCsGsCsACsC 199 (Sp, Sp, Sp, Sp, Sp, Rp, Rp,Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 121Gs1Cs1Cs1Us1Cs1As1Gs1Us1Cs1Us1Gs1Cs1Us1Us1Cs1Gs1CsACs 1C 200 (Sp, Sp,Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S122 Gs8Cs8Cs8Us8Cs8As8Gs8Us8Cs8Us8Gs8Cs8Us8Us8Cs8Gs8Cs1AC s8C 201 (Sp,Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp)-5S-9R-4S 123 Gs9Cs9Cs9Us9Cs9As9Gs9Us9Cs9Us9Gs9Cs9Us9Us9Cs9Gs9Cs1AC s9C202 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp, Rp, Rp, Sp, Sp, Sp,Sp)- 5S-9R-4S 124 Gs10Cs10Cs10Us10Cs10As10Gs10Us10Cs10Us10Gs10Cs10Us10Us10Cs10Gs10Cs1ACs10C 203 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp,Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 125Gs11Cs11Cs11Us11Cs11As11Gs11Us11Cs11Us11Gs11Cs11Us11Us11Cs11Gs11Cs1ACs11C 204 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp,Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 126Gs12Cs12Cs12Us12Cs12As12Gs12Us12Cs12Us12Gs12Cs12Us12Us12Cs12Gs12Cs1ACs12C 205 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp,Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 127Gs13Cs13Cs13Us13Cs13As13Gs13Us13Cs13Us13Gs13Cs13Us13Us13Cs13Gs13Cs1ACs13C 206 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp,Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 128Gs14Cs14Cs14Us14Cs14As14Gs14Us14Cs14Us14Gs14Cs14Us14Us14Cs14Gs14Cs1ACs14C 207 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp Rp, Rp,Rp, Rp, Sp, Sp, Sp, Sp)- 5S-9R-4S 129Gs15Cs15Cs15Us15Cs15As15Gs15Us15Cs15Us15Gs15Cs15Us15Us15Cs15Gs15Cs1ACs15C

In some embodiments, the present disclosure provides oligonucleotidesand/or oligonucleotide compositions that are useful for treatingHuntington's disease, for example, selected from:

TABLE N1 Example sequences targeting rs362307 SEQ ID NO: WV-904G*G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T rs362307 P13 130 WV-905G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C rs362307 P12 131 WV-906G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C*C rs362307 P11 132 WV-907C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C*C*A rs362307 P10 133 WV-908A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C*C*A*A rs362307 P9 134 WV-909C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C*C*A*A*A rs362307 P8 135 WV-910mG*mG*mG*mC*mA*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T rs362307 P13 136 WV-911mG*mG*mC*mA*mC*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C rs362307 P12 137 WV-912mG*mC*mA*mC*mA*A*G*G*G*C*A*C*A*G*A*C*T*T*C*C rs362307 P11 138 WV-913mC*mA*mC*mA*mA*G*G*G*C*A*C*A*G*A*C*T*T*C*C*A rs362307 P10 139 WV-914mA*mC*mA*mA*mG*G*G*C*A*C*A*G*A*C*T*T*C*C*A*A rs362307 P9 140 WV-915mC*mA*mA*mG*mG*G*C*A*C*A*G*A*C*T*T*C*C*A*A*A rs362307 P8 141 WV-916mG*mG*mG*mC*mA*C*A*A*G*G*G*C*A*C*A*mG*mA*mC*mU*mU rs362307 P13 142WV-917 mG*mG*mC*mA*mC*A*A*G*G*G*C*A*C*A*G*mA*mC*mU*mU*mC rs362307 P12143 WV-918 mG*mC*mA*mC*mA*A*G*G*G*C*A*C*A*G*A*mC*mU*mU*mC*mC rs362307P11 144 WV-919 mC*mA*mC*mA*mA*G*G*G*C*A*C*A*G*A*C*mU*mU*mC*mC*mArs362307 P10 145 WV-920mA*mC*mA*mA*mG*G*G*C*A*C*A*G*A*C*T*mU*mC*mC*mA*mA rs362307 P9 146 WV-921mC*mA*mA*mG*mG*G*C*A*C*A*G*A*C*T*T*mC*mC*mA*mA*mA rs362307 P8 147 WV-922mG*mC*mA*mC*mA*mA*mG*mG*G*C*A*C*A*G*A*mC*mU*mU*mC*mC rs362307 P11 148WV-923 mC*mA*mC*mA*mA*mG*mG*G*C*A*C*A*G*A*mC*mU*mU*mC*mC*mA rs362307 P10149 WV-924 mA*mC*mA*mA*mG*mG*G*C*A*C*A*G*A*mC*mU*mU*mC*mC*mA*mA rs362307P9 150 WV-925 mC*mA*mA*mG*mG*G*C*A*C*A*G*A*mC*mU*mU*mC*mC*mA*mA*mArs362307 P8 151 WV-926 mGmCmAmCmAmAmGmG*G*C*A*C*A*G*A*mCmUmUmCmCrs362307 P11 152 WV-927 mCmAmCmAmAmGmG*G*C*A*C*A*G*A*mCmUmUmCmCmArs362307 P10 153 WV-928 mAmCmAmAmGmG*G*C*A*C*A*G*A*mCmUmUmCmCmAmArs362307 P9 154 WV-929 mCmAmAmGmG*G*C*A*C*A*G*A*mCmUmUmCmCmAmAmArs362307 P8 155 WV-930 mGmGmGmCmA*C*A*A*G*G*G*C*A*C*A*mGmAmCmUmUrs362307 P13 156 WV-931 mGmGmCmAmC*A*A*G*G*G*C*A*C*A*G*mAmCmUmUmCrs362307 P12 157 WV-932 mGmCmAmCmA*A*G*G*G*C*A*C*A*G*A*mCmUmUmCmCrs362307 P11 158 WV-933 mCmAmCmAmA*G*G*G*C*A*C*A*G*A*C*mUmUmCmCmArs362307 P10 159 WV-934 mAmCmAmAmG*G*G*C*A*C*A*G*A*C*T*mUmCmCmAmArs362307 P9 160 WV-935 mCmAmAmGmG*G*C*A*C*A*G*A*C*T*T*mCmCmAmAmArs362307 P8 161 WV-936G*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST rs362307 P13162 WV-937 G*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SCrs362307 P12 163 WV-938G*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC rs362307 P11164 WV-939 C*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SArs362307 P10 165 WV-940A*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA rs362307 P9166 WV-941 C*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA*SArs362307 P8 167 WV-1085mG*SmG*SmC*SmA*SmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmA*SmC* rs362307 P12168 SmU*SmU*SmC WV-1086mG*RmG*RmC*RmA*RmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmA*RmC rs362307 P12169 *RmU*RmU*RmC WV-1087mGmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmUmC rs362307 P12 170WV-1088 mG*SmG*SmC*SmA*SmC*SmA*SmA*SmG*SG*SG*SC*SA*SC*RA*SG*SA*Srs362307 P12 171 C*ST*ST*SC WV-1089mG*RmG*RmC*RmA*RmC*RmA*RmA*RmG*SG*SG*SC*SA*SC*RA*SG*SA* rs362307 P12 172SC*ST*ST*SC WV-1090 mGmGmCmAmCmAmAmG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SCrs362307 P12 173 WV-1091mG*RmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*R rs362307 P12 174mC WV-1092 mG*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*Srs362307 P12 175 mC WV-982G*SC*SA*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA rs362307 P16176 WV-983 C*SA*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SCrs362307 P15 177 WV-984A*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST rs362307 P14178 WV-985 A*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA*SA*SGrs362307 P7 179 WV-986A*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA*SA*SG*SG rs362307 P6180 WV-987 G*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA*SA*SG*SG*SCrs362307 P5 181 WV-1234mG*mG*mC*mA*mC*A*A*G*G*G*C*A*C*A*G*mA*mC*mU*BrdU*mC rs362307 P12 182WV-1235 mG*mG*mC*mA*mC*A*A*G*G*G*C*A*C*A*G*mA*mC*BrdU*BrdU*mC rs362307P12 183 WV-1067 G*G*G*C*A*C*A*A*G*G*G*C*d2AP*C*A*G*A*C*T*T rs362307 P13184 WV-1068 G*G*C*A*C*A*A*G*G*G*C*d2AP*C*A*G*A*C*T*T*C rs362307 P12 185WV-1069 G*C*A*C*A*A*G*G*G*C*d2AP*C*A*G*A*C*T*T*C*C rs362307 P11 186WV-1070 G*G*G*C*A*C*A*A*G*G*G*C*dDAP*C*A*G*A*C*T*T rs362307 P13 187WV-1071 G*G*C*A*C*A*A*G*G*G*C*dDAP*C*A*G*A*C*T*T*C rs362307 P12 188WV-1072 G*C*A*C*A*A*G*G*G*C*dDAP*C*A*G*A*C*T*T*C*C rs362307 P11 189WV-1510 G*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*SC rs362307P12 190 WV-1511 G*mGmCmAmC*A*A*G*G*G*C*A*C*A*G*mAmCmUmU*C rs362307 P12191 WV-1497 mG*mGmCmAmC*A*A*G*G*G*C*A*C*A*G*mAmCmUmU*mC rs362307 P12 192WV-1655 Geo*Geom5CeoAeom5Ceo*A*A*G*G*G*C*A*C*A*G*Aeom5CeoTeoTeo*m5Ceors362307 P12 193

TABLE N2 Example sequences targeting rs362306 WV-1001G*A*G*C*A*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A rs362306 P10 194 WV-1002A*G*C*A*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C rs362306 P9 195 WV-1003G*C*A*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C*A rs362306 P8 196 WV-1004C*A*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C*A*A rs362306 P7 197 WV-1005A*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C*A*A*C rs362306 P6 198 WV-1006G*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C*A*A*C*C rs362306 P5 199 WV-1007mG*mA*mG*mC*mA*G*C*T*G*C*A*A*C*C*T*G*G*C*A*A rs362306 P10 200 WV-1008mA*mG*mC*mA*mG*C*T*G*C*A*A*C*C*T*G*G*C*A*A*C rs362306 P9 201 WV-1009mG*mC*mA*mG*mC*T*G*C*A*A*C*C*T*G*G*C*A*A*C*A rs362306 P8 202 WV-1010mC*mA*mG*mC*mU*G*C*A*A*C*C*T*G*G*C*A*A*C*A*A rs362306 P7 203 WV-1011mA*mG*mC*mU*mG*C*A*A*C*C*T*G*G*C*A*A*C*A*A*C rs362306 P6 204 WV-1012mG*mC*mU*mG*mC*A*A*C*C*T*G*G*C*A*A*C*A*A*C*C rs362306 P5 205 WV-1013mG*mA*mG*mC*mA*G*C*T*G*C*A*A*C*C*T*mG*mG*mC*mA*mA rs362306 P10 206WV-1014 mA*mG*mC*mA*mG*C*T*G*C*A*A*C*C*T*G*mG*mC*mA*mA*mC rs362306 P9207 WV-1015 mG*mC*mA*mG*mC*T*G*C*A*A*C*C*T*G*G*mC*mA*mA*mC*mA rs362306P8 208 WV-1016 mC*mA*mG*mC*mU*G*C*A*A*C*C*T*G*G*C*mA*mA*mC*mA*mArs362306 P7 209 WV-1017mA*mG*mC*mU*mG*C*A*A*C*C*T*G*G*C*A*mA*mC*mA*mA*mC rs362306 P6 210WV-1018 mG*mC*mU*mG*mC*A*A*C*C*T*G*G*C*A*A*mC*mA*mA*mC*mC rs362306 P5211 WV-1019 mG*mA*mG*mC*mA*mG*mC*T*G*C*A*A*C*C*mU*mG*mG*mC*mA*mArs362306 P10 212 WV-1020 mGmAmGmCmAmGmC*T*G*C*A*A*C*C*mUmGmGmCmAmArs362306 P10 213 WV-1021mA*mG*mC*mA*mG*mC*T*G*C*A*A*C*C*T*G*mG*mC*mA*mA*mC rs362306 P9 214WV-1022 mAmGmCmAmGmC*T*G*C*A*A*C*C*T*G*mGmCmAmAmC rs362306 P9 215WV-1023 mG*mC*mA*mG*mC*T*G*C*A*A*C*C*mU*mG*mG*mC*mA*mA*mC*mA rs362306 P8216 WV-1024 mGmCmAmGmC*T*G*C*A*A*C*C*mUmGmGmCmAmAmCmA rs362306 P8 217WV-1025 mGmAmGmCmA*G*C*T*G*C*A*A*C*C*T*mGmGmCmAmA rs362306 P10 218WV-1026 mAmGmCmAmG*C*T*G*C*A*A*C*C*T*G*mGmCmAmAmC rs362306 P9 219WV-1027 mGmCmAmGmC*T*G*C*A*A*C*C*T*G*G*mCmAmAmCmA rs362306 P8 220WV-1028 mCmAmGmCmU*G*C*A*A*C*C*T*G*G*C*mAmAmCmAmA rs362306 P7 221WV-1029 mAmGmCmUmG*C*A*A*C*C*T*G*G*C*A*mAmCmAmAmC rs362306 P6 222WV-1030 mGmCmUmGmC*A*A*C*C*T*G*G*C*A*A*mCmAmAmCmC rs362306 P5 223 WV-952G*SA*SG*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA rs362306 P10224 WV-953 A*SG*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SCrs362306 P9 225 WV-954G*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SC*SA rs362306 P8226 WV-955 C*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SC*SA*SArs362306 P7 227 WV-956A*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SC*SA*SA*SC rs362306 P6228 WV-957 G*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SC*SA*SA*SC*SCrs362306 P5 229

TABLE N3 Example sequences targeting rs362268 WV-1031G*G*G*C*C*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A rs362268 P10 230 WV-1032G*G*C*C*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G rs362268 P9 231 WV-1033G*C*C*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G*G rs362268 P8 232 WV-1034C*C*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G*G*A rs362268 P7 233 WV-1035C*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G*G*A*G rs362268 P6 234 WV-1036A*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G*G*A*G*G rs362268 P5 235 WV-1037mG*mG*mG*mC*mC*A*A*C*A*G*C*C*A*G*C*C*T*G*C*A rs362268 P10 236 WV-1038mG*mG*mC*mC*mA*A*C*A*G*C*C*A*G*C*C*T*G*C*A*G rs362268 P9 237 WV-1039mG*mC*mC*mA*mA*C*A*G*C*C*A*G*C*C*T*G*C*A*G*G rs362268 P8 238 WV-1040mC*mC*mA*mA*mC*A*G*C*C*A*G*C*C*T*G*C*A*G*G*A rs362268 P7 239 WV-1041mC*mA*mA*mC*mA*G*C*C*A*G*C*C*T*G*C*A*G*G*A*G rs362268 P6 240 WV-1042mA*mA*mC*mA*mG*C*C*A*G*C*C*T*G*C*A*G*G*A*G*G rs362268 P5 241 WV-1043mG*mG*mG*mC*mC*A*A*C*A*G*C*C*A*G*C*mC*mU*mG*mC*mA rs362268 P10 242WV-1044 mG*mG*mC*mC*mA*A*C*A*G*C*C*A*G*C*C*mU*mG*mC*mA*mG rs362268 P9243 WV-1045 mG*mC*mC*mA*mA*C*A*G*C*C*A*G*C*C*T*mG*mC*mA*mG*mG rs362268P8 244 WV-1046 mC*mC*mA*mA*mC*A*G*C*C*A*G*C*C*T*G*mC*mA*mG*mG*mArs362268 P7 245 WV-1047mC*mA*mA*mC*mA*G*C*C*A*G*C*C*T*G*C*mA*mG*mG*mA*mG rs362268 P6 246WV-1048 mA*mA*mC*mA*mG*C*C*A*G*C*C*T*G*C*A*mG*mG*mA*mG*mG rs362268 P5247 WV-1049 mG*mG*mG*mC*mC*mA*mA*C*A*G*C*C*A*G*mC*mC*mU*mG*mC*mArs362268 P10 248 WV-1050 mGmGmGmCmCmAmA*C*A*G*C*C*A*G*mCmCmUmGmCmArs362268 P10 249 WV-1051mG*mG*mC*mC*mA*mA*C*A*G*C*C*A*G*C*C*mU*mG*mC*mA*mG rs362268 P9 250WV-1052 mGmGmCmCmAmA*C*A*G*C*C*A*G*C*C*mUmGmCmAmG rs362268 P9 251WV-1053 mG*mC*mC*mA*mA*C*A*G*C*C*A*G*mC*mC*mU*mG*mC*mA*mG*mG rs362268 P8252 WV-1054 mGmCmCmAmA*C*A*G*C*C*A*G*mCmCmUmGmCmAmGmG rs362268 P8 253WV-1055 mGmGmGmCmC*A*A*C*A*G*C*C*A*G*C*mCmUmGmCmA rs362268 P10 254WV-1056 mGmGmCmCmA*A*C*A*G*C*C*A*G*C*C*mUmGmCmAmG rs362268 P9 255WV-1057 mGmCmCmAmA*C*A*G*C*C*A*G*C*C*T*mGmCmAmGmG rs362268 P8 256WV-1058 mCmCmAmAmC*A*G*C*C*A*G*C*C*T*G*mCmAmGmGmA rs362268 P7 257WV-1059 mCmAmAmCmA*G*C*C*A*G*C*C*T*G*C*mAmGmGmAmG rs362268 P6 258WV-1060 mAmAmCmAmG*C*C*A*G*C*C*T*G*C*A*mGmGmAmGmG rs362268 P5 259 WV-960G*SG*SG*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA rs362268 P10260 WV-961 G*SG*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SGrs362268 P9 261 WV-962G*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SG*SG rs362268 P8262 WV-963 C*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SG*SG*SArs362268 P7 263 WV-964C*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SG*SG*SA*SG rs362268 P6264 WV-965 A*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SG*SG*SA*SG*SGrs362268 P5 265

TABLE N4 Example sequences targeting rs7685686 ONT-450A*T*T*A*A*T*A*A*A*T*T*G*T*C*A*T*C*A*C*C rs7685686 P13 266 ONT-451A*ST*ST*SA*SA*ST*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SC*SA*SC*SC rs7685686 P13267 ONT-452 A*ST*ST*SA*SA*ST*SA*SA*SA*ST*ST*SG*ST*SC*SA*RT*SC*SA*SC*SCrs7685686 P13 268 WV-1077mA*SmU*SmU*SmA*SmA*SmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmC* rs7685686 P13269 SmA*SmC*SmC WV-1078mA*RmU*RmU*RmA*RmA*RmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmC rs7685686 P13270 *RmA*RmC*RmC WV-1079mA*SmU*SmU*SmA*SmA*SmU*SmA*SmA*SA*ST*ST*SG*ST*SC*RA*ST*SC rs7685686 P13271 *SA*SC*SC WV-1080mA*RmU*RmU*RmA*RmA*RmU*RmA*RmA*SA*ST*ST*SG*ST*SC*RA*ST* rs7685686 P13272 SC*SA*SC*SC WV-1081mAmUmUmAmAmUmAmA*SA*ST*ST*SG*ST*SC*RA*ST*SC*SA*SC*SC rs7685686 P13 273WV-1082 mAmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmCmAmCmC rs7685686P13 274 WV-1083 mA*SmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmCmAmC*SmCrs7685686 P13 275 WV-1084mA*RmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmCmAmC*RmC rs7685686 P13276 WV-1508 A*SmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SmCmAmC*SCrs7685686 P13 277 WV-1509 A*mUmUmAmAmU*A*A*A*T*T*G*T*C*A*T*mCmAmC*Crs7685686 P13 278 WV-2023 T*G*T*C*A*T*C*A*C*C*A*G*A*A*A*mA*mA*mG*mU*mCrs7685686 P3 279 WV-2024 mU*T*G*T*C*A*T*C*A*C*C*A*G*A*A*mA*mA*mA*mG*mUrs7685686 P4 280 WV-2025 T*T*G*T*C*A*T*C*A*C*C*A*G*A*A*mA*mA*mA*mG*mUrs7685686 P4 281 WV-2026 mA*mU*T*G*T*C*A*T*C*A*C*C*A*G*A*mA*mA*mA*mA*mGrs7685686 P5 282 WV-2027 mA*T*T*G*T*C*A*T*C*A*C*C*A*G*A*mA*mA*mA*mA*mGrs7685686 P5 283 WV-2028 mA*mA*mU*T*G*T*C*A*T*C*A*C*C*A*G*mA*mA*mA*mA*mArs7685686 P6 284 WV-2029 mA*mA*T*T*G*T*C*A*T*C*A*C*C*A*G*mA*mA*mA*mA*mArs7685686 P6 285 WV-2030 mA*mA*mA*T*T*G*T*C*A*T*C*A*C*C*A*mG*mA*mA*mA*mArs7685686 P7 286 WV-2031mA*mA*mA*mU*T*G*T*C*A*T*C*A*C*C*A*mG*mA*mA*mA*mA rs7685686 P7 287WV-2032 mU*mA*mA*mA*mU*T*G*T*C*A*T*C*A*C*C*A*mG*mA*mA*mA rs7685686 P8288 WV-2033 mU*mA*mA*mA*mU*T*G*T*C*A*T*C*A*C*C*mA*mG*mA*mA*mA rs7685686P8 289 WV-2034 mA*mU*mA*mA*mA*T*T*G*T*C*A*T*C*A*C*C*mA*mG*mA*mArs7685686 P9 290 WV-2035mA*mU*mA*mA*mA*T*T*G*T*C*A*T*C*A*C*mC*mA*mG*mA*mA rs7685686 P9 291WV-2036 mA*mA*mU*mA*mA*A*T*T*G*T*C*A*T*C*A*C*C*mA*mG*mA rs7685686 P10292 WV-2037 mA*mA*mU*mA*mA*A*T*T*G*T*C*A*T*C*A*C*mC*mA*mG*mA rs7685686P10 293 WV-2038 mA*mA*mU*mA*mA*A*T*T*G*T*C*A*T*C*A*mC*mC*mA*mG*mArs7685686 P10 294 WV-2039 mU*mA*mA*mU*mA*A*A*T*T*G*T*C*A*T*C*A*C*C*mA*mGrs7685686 P11 295 WV-2040mU*mA*mA*mU*mA*A*A*T*T*G*T*C*A*T*C*A*C*mC*mA*mG rs7685686 P11 296WV-2041 mU*mA*mA*mU*mA*A*A*T*T*G*T*C*A*T*C*A*mC*mC*mA*mG rs7685686 P11297 WV-2042 mU*mA*mA*mU*mA*A*A*T*T*G*T*C*A*T*C*mA*mC*mC*mA*mG rs7685686P11 298 WV-2043 mU*mU*mA*mA*mU*A*A*A*T*T*G*T*C*A*T*C*A*C*C*mA rs7685686P12 299 WV-2044 mU*mU*mA*mA*mU*A*A*A*T*T*G*T*C*A*T*C*A*C*mC*mA rs7685686P12 300 WV-2045 mU*mU*mA*mA*mU*A*A*A*T*T*G*T*C*A*T*C*A*mC*mC*mArs7685686 P12 301 WV-2046mU*mU*mA*mA*mU*A*A*A*T*T*G*T*C*A*T*C*mA*mC*mC*mA rs7685686 P12 302WV-2047 mA*mU*mU*mA*mA*T*A*A*A*T*T*G*T*C*A*T*C*A*C*C rs7685686 P13 303WV-2048 mA*mU*mU*mA*mA*T*A*A*A*T*T*G*T*C*A*T*C*A*C*mC rs7685686 P13 304WV-2049 mA*mU*mU*mA*mA*T*A*A*A*T*T*G*T*C*A*T*C*A*mC*mC rs7685686 P13 305WV-2050 mA*mU*mU*mA*mA*T*A*A*A*T*T*G*T*C*A*T*C*mA*mC*mC rs7685686 P13306 WV-2051 mU*mA*mU*mU*mA*A*T*A*A*A*T*T*G*T*C*A*T*C*A*C rs7685686 P14307 WV-2052 mU*mA*mU*mU*mA*A*T*A*A*A*T*T*G*T*C*A*T*C*A*mC rs7685686 P14308 WV-2053 mU*mA*mU*mU*mA*A*T*A*A*A*T*T*G*T*C*A*T*C*mA*mC rs7685686 P14309 WV-2054 mC*mU*mA*mU*mU*A*A*T*A*A*A*T*T*G*T*C*A*T*C*A rs7685686 P15310 WV-2055 mC*mU*mA*mU*mU*A*A*T*A*A*A*T*T*G*T*C*A*T*C*mA rs7685686 P15311 WV-2056 mA*mC*mU*mA*mU*T*A*A*T*A*A*A*T*T*G*T*C*A*T*C rs7685686 P16312 WV-2057 T*G*T*C*A*T*C*A*C*C*A*G*A*A*A*mAmAmGmU*mC rs7685686 P3 313WV-2058 mU*T*G*T*C*A*T*C*A*C*C*A*G*A*A*mAmAmAmG*mU rs7685686 P4 314WV-2059 T*T*G*T*C*A*T*C*A*C*C*A*G*A*A*mAmAmAmG*mU rs7685686 P4 315WV-2060 mA*mU*T*G*T*C*A*T*C*A*C*C*A*G*A*mAmAmAmA*mG rs7685686 P5 316WV-2061 mA*T*T*G*T*C*A*T*C*A*C*C*A*G*A*mAmAmAmA*mG rs7685686 P5 317WV-2062 mA*mAmU*T*G*T*C*A*T*C*A*C*C*A*G*mAmAmAmA*mA rs7685686 P6 318WV-2063 mA*mA*T*T*G*T*C*A*T*C*A*C*C*A*G*mAmAmAmA*mA rs7685686 P6 319WV-2064 mA*mAmA*T*T*G*T*C*A*T*C*A*C*C*A*mGmAmAmA*mA rs7685686 P7 320WV-2065 mA*mAmAmU*T*G*T*C*A*T*C*A*C*C*A*mGmAmAmA*mA rs7685686 P7 321WV-2066 mU*mAmAmAmU*T*G*T*C*A*T*C*A*C*C*A*mGmAmA*mA rs7685686 P8 322WV-2067 mU*mAmAmAmU*T*G*T*C*A*T*C*A*C*C*mAmGmAmA*mA rs7685686 P8 323WV-2068 mA*mUmAmAmA*T*T*G*T*C*A*T*C*A*C*C*mAmGmA*mA rs7685686 P9 324WV-2069 mA*mUmAmAmA*T*T*G*T*C*A*T*C*A*C*mCmAmGmA*mA rs7685686 P9 325WV-2070 mA*mAmUmAmA*A*T*T*G*T*C*A*T*C*A*C*C*mAmG*mA rs7685686 P10 326WV-2071 mA*mAmUmAmA*A*T*T*G*T*C*A*T*C*A*C*mCmAmG*mA rs7685686 P10 327WV-2072 mA*mAmUmAmA*A*T*T*G*T*C*A*T*C*A*mCmCmAmG*mA rs7685686 P10 328WV-2073 mU*mAmAmUmA*A*A*T*T*G*T*C*A*T*C*A*C*C*mA*mG rs7685686 P11 329WV-2074 mU*mAmAmUmA*A*A*T*T*G*T*C*A*T*C*A*C*mCmA*mG rs7685686 P11 330WV-2075 mU*mAmAmUmA*A*A*T*T*G*T*C*A*T*C*A*mCmCmA*mG rs7685686 P11 331WV-2076 mU*mAmAmUmA*A*A*T*T*G*T*C*A*T*C*mAmCmCmA*mG rs7685686 P11 332WV-2077 mU*mUmAmAmU*A*A*A*T*T*G*T*C*A*T*C*A*C*C*mA rs7685686 P12 333WV-2078 mU*mUmAmAmU*A*A*A*T*T*G*T*C*A*T*C*A*C*mC*mA rs7685686 P12 334WV-2079 mU*mUmAmAmU*A*A*A*T*T*G*T*C*A*T*C*A*mCmC*mA rs7685686 P12 335WV-2080 mU*mUmAmAmU*A*A*A*T*T*G*T*C*A*T*C*mAmCmC*mA rs7685686 P12 336WV-2081 mA*mUmUmAmA*T*A*A*A*T*T*G*T*C*A*T*C*A*C*C rs7685686 P13 337WV-2082 mA*mUmUmAmA*T*A*A*A*T*T*G*T*C*A*T*C*A*C*mC rs7685686 P13 338WV-2083 mA*mUmUmAmA*T*A*A*A*T*T*G*T*C*A*T*C*A*mC*mC rs7685686 P13 339WV-2084 mA*mUmUmAmA*T*A*A*A*T*T*G*T*C*A*T*C*mAmC*mC rs7685686 P13 340WV-2085 mU*mAmUmUmA*A*T*A*A*A*T*T*G*T*C*A*T*C*A*C rs7685686 P14 341WV-2086 mU*mAmUmUmA*A*T*A*A*A*T*T*G*T*C*A*T*C*A*mC rs7685686 P14 342WV-2087 mU*mAmUmUmA*A*T*A*A*A*T*T*G*T*C*A*T*C*mA*mC rs7685686 P14 343WV-2088 mC*mUmAmUmU*A*A*T*A*A*A*T*T*G*T*C*A*T*C*A rs7685686 P15 344WV-2089 mC*mUmAmUmU*A*A*T*A*A*A*T*T*G*T*C*A*T*C*mA rs7685686 P15 345WV-2090 mA*mCmUmAmU*T*A*A*T*A*A*A*T*T*G*T*C*A*T*C rs7685686 P16 346

TABLE N1 Example sequences targeting rs362307 - continued (certainfeatures) WV-904 All DNA, stereorandom PS rs362307 P13 WV-905 All DNA,stereorandom PS rs362307 P12 WV-906 All DNA, stereorandom PS rs362307P11 WV-907 All DNA, stereorandom PS rs362307 P10 WV-908 All DNA,stereorandom PS rs362307 P9 WV-909 All DNA, stereorandom PS rs362307 P8WV-910 5-15 (2′-OMe-DNA), stereorandom PS rs362307 P13 WV-911 5-15(2′-OMe-DNA), stereorandom PS rs362307 P12 WV-912 5-15 (2′-OMe-DNA),stereorandom PS rs362307 P11 WV-913 5-15 (2′-OMe-DNA), stereorandom PSrs362307 P10 WV-914 5-15 (2′-OMe-DNA), stereorandom PS rs362307 P9WV-915 5-15 (2′-OMe-DNA), stereorandom PS rs362307 P8 WV-916 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P13 WV-917 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P12 WV-918 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P11 WV-919 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P10 WV-920 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P9 WV-921 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P8 WV-922 8-7-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P11 WV-923 7-7-6(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P10 WV-924 6-7-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P9 WV-925 5-7-8(2′-OMe-DNA-2′-OMe), stereorandom PS rs362307 P8 WV-926 8-7-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362307 P11WV-927 7-7-6 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362307 P10 WV-928 6-7-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO inthe wings rs362307 P9 WV-929 5-7-8 (2′-OMe-DNA-2′-OMe), stereorandom PS,PO in the wings rs362307 P8 WV-930 5-10-5 (2′-OMe-DNA-2′-OMe),stereorandom PS, PO in the wings rs362307 P13 WV-931 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362307 P12WV-932 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362307 P11 WV-933 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO inthe wings rs362307 P10 WV-934 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandomPS, PO in the wings rs362307 P9 WV-935 5-10-5 (2′-OMe-DNA-2′-OMe),stereorandom PS, PO in the wings rs362307 P8 WV-936 All DNA, stereopure,One Rp rs362307 P13 WV-937 All DNA, stereopure, One Rp rs362307 P12WV-938 All DNA, stereopure, One Rp rs362307 P11 WV-939 All DNA,stereopure, One Rp rs362307 P10 WV-940 All DNA, stereopure, One Rprs362307 P9 WV-941 All DNA, stereopure, One Rp rs362307 P8 WV-10855-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA rs362307P12 WV-1086 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNAand Rp wings rs362307 P12 WV-1087 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer,Stereopure, One Rp in DNA, PO wings rs362307 P12 WV-1088 8-12(2′-OMe-DNA) hemimer, Stereopure, One Rp in DNA, Sp wing rs362307 P12WV-1089 8-12 (2′-OMe-DNA) hemimer, Stereopure, One Rp in DNA and Rp wingrs362307 P12 WV-1090 8-12 (2′-OMe-DNA) hemimer, Srereopure, One Rp inDNA and PO wing rs362307 P12 WV-1091 5-10-5 (2′-OMe-DNA-2′-OMe) gapmer,Stereopure, One Rp in DNA, rs362307 P12 First and last PS as Rp and restPO wing WV-1092 5-10-5 (2′-OMe-DNA-2′-OMe) gapmer, Stereopure, One Rp inDNA, rs362307 P12 First and last PS as Sp and rest PO wing WV-982 AllDNA, stereopure, One Rp rs362307 P16 WV-983 All DNA, stereopure, One Rprs362307 P15 WV-984 All DNA, stereopure, One Rp rs362307 P14 WV-985 AllDNA, stereopure, One Rp rs362307 P7 WV-986 All DNA, stereopure, One Rprs362307 P6 WV-987 All DNA, stereopure, One Rp rs362307 P5 WV-12345-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereorandom, One Br-dU rs362307 P12WV-1235 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereorandom, Two Br-dUrs362307 P12 WV-1067 All DNA, stereorandom PS, one 2-amino puriners362307 P13 WV-1068 All DNA, stereorandom PS, one 2-amino puriners362307 P12 WV-1069 All DNA, stereorandom PS, one 2-amino puriners362307 P11 WV-1070 All DNA, stereorandom PS, one 2,6-diamino puriners362307 P13 WV-1071 All DNA, stereorandom PS, one 2,6-diamino puriners362307 P12 WV-1072 All DNA, stereorandom PS, one 2,6-diamino puriners362307 P11 WV-1510 1-4-10-4-1 (DNA/2′-OMe) gapmer, Stereopure, one Rpin the DNA, rs362307 P12 first and last nucletotide is DNA and first andlast PS are Sp WV-1511 1-4-10-4-1 (DNA/2′-OMe) gapmer, Stereorandom, 1stand last PS, rs362307 P12 rest of the wing is PO WV-1497 5-10-5(2′-OMe-DNA-2′-OMe) gapmer, Stereorandom, First and rs362307 P12 last PSand rest PO wing WV-1655 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer,Stereorandom, PO wings rs362307 P12 with One PS on each end

TABLE N2 Example sequences targeting rs362306 - continued (certainfeatures) WV-1001 All DNA, stereorandom PS rs362306 P10 WV-1002 All DNA,stereorandom PS rs362306 P9 WV-1003 All DNA, stereorandom PS rs362306 P8WV-1004 All DNA, stereorandom PS rs362306 P7 WV-1005 All DNA,stereorandom PS rs362306 P6 WV-1006 All DNA, stereorandom PS rs362306 P5WV-1007 5-15 (2′-OMe-DNA), stereorandom PS rs362306 P10 WV-1008 5-15(2′-OMe-DNA), stereorandom PS rs362306 P9 WV-1009 5-15 (2′-OMe-DNA),stereorandom PS rs362306 P8 WV-1010 5-15 (2′-OMe-DNA), stereorandom PSrs362306 P7 WV-1011 5-15 (2′-OMe-DNA), stereorandom PS rs362306 P6WV-1012 5-15 (2′-OMe-DNA), stereorandom PS rs362306 P5 WV-1013 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P10 WV-1014 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P9 WV-1015 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P8 WV-1016 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P7 WV-1017 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P6 WV-1018 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P5 WV-1019 7-7-6(2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P10 WV-1020 7-7-6(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in wings rs362306 P10 WV-10216-7-5 (2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P9 WV-1022 6-7-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362306 P9WV-1023 5-7-8 (2′-OMe-DNA-2′-OMe), stereorandom PS rs362306 P8 WV-10245-7-8 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362306 P8WV-1025 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362306 P10 WV-1026 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO inthe wings rs362306 P9 WV-1027 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandomPS, PO in the wings rs362306 P8 WV-1028 5-10-5 (2′-OMe-DNA-2′-OMe),stereorandom PS, PO in the wings rs362306 P7 WV-1029 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362306 P6WV-1030 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362306 P5 WV-952 All DNA, stereopure, One Rp rs362306 P10 WV-953 AllDNA, stereopure, One Rp rs362306 P9 WV-954 All DNA, stereopure, One Rprs362306 P8 WV-955 All DNA, stereopure, One Rp rs362306 P7 WV-956 AllDNA, stereopure, One Rp rs362306 P6 WV-957 All DNA, stereopure, One Rprs362306 P5

TABLE N3 Example sequences targeting rs362268 - continued (certainfeatures) WV-1031 All DNA, stereorandom PS rs362268 P10 WV-1032 All DNA,stereorandom PS rs362268 P9 WV-1033 All DNA, stereorandom PS rs362268 P8WV-1034 All DNA, stereorandom PS rs362268 P7 WV-1035 All DNA,stereorandom PS rs362268 P6 WV-1036 All DNA, stereorandom PS rs362268 P5WV-1037 5-15 (2′-OMe-DNA), stereorandom PS rs362268 P10 WV-1038 5-15(2′-OMe-DNA), stereorandom PS rs362268 P9 WV-1039 5-15 (2′-OMe-DNA),stereorandom PS rs362268 P8 WV-1040 5-15 (2′-OMe-DNA), stereorandom PSrs362268 P7 WV-1041 5-15 (2′-OMe-DNA), stereorandom PS rs362268 P6WV-1042 5-15 (2′-OMe-DNA), stereorandom PS rs362268 P5 WV-1043 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P10 WV-1044 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P9 WV-1045 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P8 WV-1046 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P7 WV-1047 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P6 WV-1048 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P5 WV-1049 7-7-6(2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P10 WV-1050 7-7-6(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in wings rs362268 P10 WV-10516-7-5 (2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P9 WV-1052 6-7-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362268 P9WV-1053 5-7-8 (2′-OMe-DNA-2′-OMe), stereorandom PS rs362268 P8 WV-10545-7-8 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362268 P8WV-1055 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362268 P10 WV-1056 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO inthe wings rs362268 P9 WV-1057 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandomPS, PO in the wings rs362268 P8 WV-1058 5-10-5 (2′-OMe-DNA-2′-OMe),stereorandom PS, PO in the wings rs362268 P7 WV-1059 5-10-5(2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings rs362268 P6WV-1060 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wingsrs362268 P5 WV-960 All DNA, stereopure, One Rp rs362268 P10 WV-961 AllDNA, stereopure, One Rp rs362268 P9 WV-962 All DNA, stereopure, One Rprs362268 P8 WV-963 All DNA, stereopure, One Rp rs362268 P7 WV-964 AllDNA, stereopure, One Rp rs362268 P6 WV-965 All DNA, stereopure, One Rprs362268 P5

TABLE N4 Example sequences targeting rs7685686 - continued (certainfeatures) ONT-450 All DNA, stereorandom PS rs7685686 P13 ONT-451 AllDNA, stereopure, One Rp in DNA between position 14 and 15 rs7685686 P13ONT-452 All DNA, stereopure, One Rp in DNA between position 15 and 16rs7685686 P13 WV-1077 6-10-4 (2′-OMe-DNA-2′-OMe) Gapmer, stereopure withone rs7685686 P13 Rp in DNA between position 14 and 15 WV-1078 6-10-4(2′-OMe-DNA-2′-OMe) Gapmer, stereopure with one rs7685686 P13 Rp in DNAbetween position 14 and 15 and Rp wings WV-1079 8-12 (2′-OMe-DNA)Hemimer, stereopure with one Rp in DNA rs7685686 P13 between position 14and 15 and Sp wing WV-1080 8-12 (2′-OMe-DNA) Hemimer, stereopure withone Rp in DNA rs7685686 P13 between position 14 and 15 and Rp wingWV-1081 8-12 (2′-OMe-DNA) Hemimer, stereopure with one Rp in DNArs7685686 P13 between position 14 and 15 and PO wing WV-1082 6-10-4(2′-OMe-DNA-2′-OMe), stereopure with one Rp in rs7685686 P13 DNA betweenposition 14 and 15 and PO wings WV-1083 6-10-4 (2′-OMe-DNA-2′-OMe),stereopure with one Rp in rs7685686 P13 DNA between position 14 and 15,first and last PS Sp and rest PO wing WV-1084 6-10-4(2′-OMe-DNA-2′-OMe), stereopure with one Rp in rs7685686 P13 DNA betweenposition 14 and 15, first and last PS Rp and rest PO wing WV-15081-5-10-3-1 (DNA/2′-OMe) Gapmer, Stereopure, one Rp in the rs7685686 P13core, first and last PS is Sp, rest is PO in the wing WV-1509 1-5-10-3-1(DNA/2′-OMe) Gapmer, Stereorandom, first and last PS, rs7685686 P13 restis PO in the wing

In Table N1-N4, * only represents a stereorandom phosphorothioatelinkage; *S represents an Sp phosphorothioate linkage, *R represents anRp phosphorothioate linkage, all non-labeled linkage is a naturalphosphate linkage, m preceding a base represents 2′-OMe, d2AP representsa 2-amino purine, and dDAP represents a 2,6-diamino purine.

In some embodiments, the present disclosure provides oligonucleotidesand/or oligonucleotide compositions that are useful for treatingHuntington's disease, for example, selected from:

TABLE N1A Example sequences targeting rs362307 SEQ ID NO: WV-936G*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*S 347 A*SC*ST*ST WV-937G*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*S 348 C*ST*ST*SC WV-938G*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*S 349 T*ST*SC*SC WV-939C*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*S 350 T*SC*SC*SA WV-940A*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*S 351 C*SC*SA*SA WV-941C*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*S 352 C*SA*SA*SA WV-1085mG*SmG*SmC*SmA*SmC*SA*SA*SG*SG*SG*SC*SA*SC*RA* 353SG*SmA*SmC*SmU*SmU*SmC WV-1086mG*RmG*RmC*RmA*RmC*SA*SA*SG*SG*SG*SC*SA*SC*RA* 354SG*SmA*RmC*RmU*RmU*RmC WV-1087mGmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAm 355 CmUmUmC WV-1088mG*SmG*SmC*SmA*SmC*SmA*SmA*SmG*SG*SG*SC*SA*SC* 356 RA*SG*SA*SC*ST*ST*SCWV-1089 mG*RmG*RmC*RmA*RmC*RmA*RmA*RmG*SG*SG*SC*SA*S 357C*RA*SG*SA*SC*ST*ST*SC WV-1090mGmGmCmAmCmAmAmG*SG*SG*SC*SA*SC*RA*SG*SA*SC* 358 ST*ST*SC WV-1091mG*RmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*Sm 359 AmCmUmU*RmC WV-1092mG*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*Sm 360 AmCmUmU*SmC WV-982G*SC*SA*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*S 361 C*RA*SG*SA WV-983C*SA*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*R 362 A*SG*SA*SC WV-984A*SG*SG*SG*SC*SA*SC*SA*SA*SG*SG*SG*SC*SA*SC*RA*S 363 G*SA*SC*ST WV-985A*SA*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*S 364 A*SA*SA*SG WV-986A*SG*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*S 365 A*SA*SG*SG WV-987G*SG*SG*SC*SA*SC*RA*SG*SA*SC*ST*ST*SC*SC*SA*SA*S 366 A*SG*SG*SC WV-1510G*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmA 367 mCmUmU*SC

TABLE N2A Example sequences targeting rs362306 WV-952G*SA*SG*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*S 368 G*SC*SA*SA WV-953A*SG*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*S 369 C*SA*SA*SC WV-954G*SC*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*S 370 A*SA*SC*SA WV-955C*SA*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*S 371 A*SC*SA*SA WV-956A*SG*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*S 372 C*SA*SA*SC WV-957G*SC*ST*SG*SC*SA*RA*SC*SC*ST*SG*SG*SC*SA*SA*SC*S 373 A*SA*SC*SC

TABLE N3A Example sequences targeting rs362268 WV-960G*SG*SG*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*S 374 T*SG*SC*SA WV-961G*SG*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*S 375 G*SC*SA*SG WV-962G*SC*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*S 376 C*SA*SG*SG WV-963C*SC*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*S 377 A*SG*SG*SA WV-964C*SA*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*S 378 G*SG*SA*SG WV-965A*SA*SC*SA*SG*SC*RC*SA*SG*SC*SC*ST*SG*SC*SA*SG*S 379 G*SA*SG*SG

TABLE N4A Example sequences targeting rs7685686 ONT-450A*T*T*A*A*T*A*A*A*T*T*G*T*C*A*T*C*A*C*C 380 ONT-451A*ST*ST*SA*SA*ST*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*SC 381 *SA*SC*SC ONT-452A*ST*ST*SA*SA*ST*SA*SA*SA*ST*ST*SG*ST*SC*SA*RT*SC 382 *SA*SC*SC WV-1077mA*SmU*SmU*SmA*SmA*SmU*SA*SA*SA*ST*ST*SG*ST*SC* 383RA*ST*SmC*SmA*SmC*SmC WV-1078mA*RmU*RmU*RmA*RmA*RmU*SA*SA*SA*ST*ST*SG*ST*S 384C*RA*ST*SmC*RmA*RmC*RmC WV-1079mA*SmU*SmU*SmA*SmA*SmU*SmA*SmA*SA*ST*ST*SG*ST* 385 SC*RA*ST*SC*SA*SC*SCWV-1080 mA*RmU*RmU*RmA*RmA*RmU*RmA*RmA*SA*ST*ST*SG*S 386T*SC*RA*ST*SC*SA*SC*SC WV-1081mAmUmUmAmAmUmAmA*SA*ST*ST*SG*ST*SC*RA*ST*SC*S 387 A*SC*SC WV-1082mAmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*Sm 388 CmAmCmC WV-1083mA*SmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*S 389 mCmAmC*SmC WV-1084mA*RmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*S 390 mCmAmC*RmC WV-1508A*SmUmUmAmAmU*SA*SA*SA*ST*ST*SG*ST*SC*RA*ST*Sm 391 CmAmC*SC

TABLE N1A Example sequences targeting rs362307 - continued WV-936 AllDNA, stereopure, One Rp WV-937 All DNA, stereopure, One Rp WV-938 AllDNA, stereopure, One Rp WV-939 All DNA, stereopure, One Rp WV-940 AllDNA, stereopure, One Rp WV-941 All DNA, stereopure, One Rp WV-10855-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA WV-10865-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA and Rpwings WV-1087 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp inDNA, PO wings WV-1088 8-12 (2′-OMe-DNA) hemimer, Stereopure, One Rp inDNA, Sp wing WV-1089 8-12 (2′-OMe-DNA) hemimer, Stereopure, One Rp inDNA and Rp wing WV-1090 8-12 (2′-OMe-DNA) hemimer, Srereopure, One Rp inDNA and PO wing WV-1091 5-10-5 (2′-OMe-DNA-2′-OMe) gapmer, Stereopure,One Rp in DNA, First and last PS as Rp and rest PO wing WV-1092 5-10-5(2′-OMe-DNA-2′-OMe) gapmer, Stereopure, One Rp in DNA, First and last PSas Sp and rest PO wing WV-982 All DNA, stereopure, One Rp WV-983 AllDNA, stereopure, One Rp WV-984 All DNA, stereopure, One Rp WV-985 AllDNA, stereopure, One Rp WV-986 All DNA, stereopure, One Rp WV-987 AllDNA, stereopure, One Rp WV-1510 1-4-10-4-1 (DNA/2′-OMe) gapmer,Stereopure, one Rp in the DNA, first and last nucletotide is DNA andfirst and last PS are Sp

TABLE N2A Example sequences targeting rs362306 - continued WV-952 AllDNA, stereopure, One Rp WV-953 All DNA, stereopure, One Rp WV-954 AllDNA, stereopure, One Rp WV-955 All DNA, stereopure, One Rp WV-956 AllDNA, stereopure, One Rp WV-957 All DNA, stereopure, One Rp

TABLE N3A Example sequences targeting rs362268 - continued WV-960 AllDNA, stereopure, One Rp WV-961 All DNA, stereopure, One Rp WV-962 AllDNA, stereopure, One Rp WV-963 All DNA, stereopure, One Rp WV-964 AllDNA, stereopure, One Rp WV-965 All DNA, stereopure, One Rp

TABLE N4A Example sequences targeting rs7685686 - continued ONT-451 AllDNA, stereopure, One Rp in DNA between position 14 and 15 ONT-452 AllDNA, stereopure, One Rp in DNA between position 15 and 16 WV-1077 6-10-4(2′-OMe-DNA-2′-OMe) Gapmer, stereopure with one Rp in DNA betweenposition 14 and 15 WV-1078 6-10-4 (2′-OMe-DNA-2′-OMe) Gapmer, stereopurewith one Rp in DNA between position 14 and 15 and Rp wings WV-1079 8-12(2′-OMe-DNA) Hemimer, stereopure with one Rp in DNA between position 14and 15 and Sp wing WV-1080 8-12 (2′-OMe-DNA) Hemimer, stereopure withone Rp in DNA between position 14 and 15 and Rp wing WV-1081 8-12(2′-OMe-DNA) Hemimer, stereopure with one Rp in DNA between position 14and 15 and PO wing WV-1082 6-10-4 (2′-OMe-DNA-2′-OMe), stereopure withone Rp in DNA between position 14 and 15 and PO wings WV-1083 6-10-4(2′-OMe-DNA-2′-OMe), stereopure with one Rp in DNA between position 14and 15, first and last PS Sp and rest PO wing WV-1084 6-10-4(2′-OMe-DNA-2′-OMe), stereopure with one Rp in DNA between position 14and 15, first and last PS Rp and rest PO wing WV-1508 1-5-10-3-1(DNA/2′-OMe) Gapmer, Stereopure, one Rp in the core, first and last PSis Sp, rest is PO in the wingIn Table N1A-N4A, * only represents a stereorandom phosphorothioatelinkage; *S represents an Sp phosphorothioate linkage, *R represents anRp phosphorothioate linkage, all non-labeled linkage is a naturalphosphate linkage, m preceding a base represents 2′-OMe, d2AP representsa 2-amino purine, and dDAP represents a 2,6-diamino purine.

In some embodiments, a provided oligonucleotide composition is achirally controlled oligonucleotide composition of an oligonucleotidetype listed in Table N1A, Table N2A, Table N3A, and Table N4A. In someembodiments, a provided composition is of WV-1092. Each oligonucleotidedescribed herein comprising a HTT sequence represents an HTToligonucleotide which was designed, constructed and tested in variousassays, for example, in vitro assays, in accordance with the presentdisclosure. For example, each HTT oligonucleotide listed in any ofTables N1A, N2A, N3A, N4A and 8, or described elsewhere herein weredesigned, constructed and tested in vitro in accordance with the presentdisclosure. Among others, every HTT oligonucleotide described herein wastested in a dual luciferase reporter assay. In some embodiments, HTToligonucleotides which were found to be particularly efficacious in thedual luciferase assay were tested in further in vitro and in vivo. Insome embodiments, a provided composition is selected from: WVE120101;WV-2603; WV-2595; WV-1510; WV-2378; and WV-2380; each of which was foundto be highly efficacious, for example, as demonstrated in vitro in thedual luciferase reporter assay in accordance with the presentdisclosure. In some embodiments, a provided composition is selectedfrom: WV-1092; WV-1497; WV-1085; WV-1086; ONT-905; and WV-2623; each ofwhich was found to be highly efficacious, for example, as demonstratedin vitro in the dual luciferase reporter assay in accordance with thepresent disclosure. Various additional HTT oligonucleotides were alsoshown to be particularly efficacious. In some embodiments, a providedcomposition is of WV-1092. In some embodiments, a provided compositionis of WV-1497. In some embodiments, a provided composition is ofWV-1085. In some embodiments, a provided composition is of WV-1086. Insome embodiments, a provided composition is of ONT-905. In someembodiments, a provided composition is of WV-2623.

The present disclosure provides compositions comprising or consisting ofa plurality of provided oligonucleotides (e.g., chirally controlledoligonucleotide compositions). In some embodiments, all such providedoligonucleotides are of the same type, i.e., all have the same basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “—XLR¹” groups in formula I).In some embodiments, all oligonucleotides of the same type areidentical. In many embodiments, however, provided compositions comprisea plurality of oligonucleotides types, typically in pre-determinedrelative amounts.

In some embodiments, a provided composition comprises a predeterminedlevel of an oligonucleotide selected from a Table. In some embodiments,a provided composition comprises a predetermined level of anoligonucleotide selected from Tables N1-N4. In some embodiments, aprovided composition comprises a predetermined level of WV-1092. In someembodiments, a provided composition comprises a predetermined level ofWV-2595. In some embodiments, a provided composition comprises apredetermined level of WV-2603. In some embodiments, a providedcomposition comprises a predetermined level of mG* SmGmCmAmC* SA* SA*SG* SG* SG* SC* SA* SC*RA* SG* SmAmCmUmU* SmC (SEQ ID NO: 1554), whereinthe oligonucleotide has a free 5′-OH and 3′-OH, m preceding a baserepresents 2′-OMe modification in the nucleoside containing the base, *Srepresents an Sp phosphorothioate linkage, *R represents an Rpphosphorothioate linkage, and all non-labeled linkage is a naturalphosphate linkage. In some embodiments, a provided composition comprisesa predetermined level of mG*SmGmGmUmC*SC*ST*SC*SC*SC*SC*SC*RA*SG*SmAmGmGmG*S mA (SEQ ID NO: 11), wherein the oligonucleotide hasa free 5′-OH and 3′-OH, m preceding a base represents 2′-OMemodification in the nucleoside containing the base, *S represents an Spphosphorothioate linkage, *R represents an Rp phosphorothioate linkage,and all non-labeled linkage is a natural phosphate linkage. In someembodiments, a provided composition comprises a predetermined level ofmG*SmUmGmCmA*SC*SA*SC*SA*SG*ST*SA*SG*RA*ST*SmGmAmGmG*S mG (SEQ ID NO:13), wherein the oligonucleotide has a free 5′-OH and 3′-OH, m precedinga base represents 2′-OMe modification in the nucleoside containing thebase, *S represents an Sp phosphorothioate linkage, *R represents an Rpphosphorothioate linkage, and all non-labeled linkage is a naturalphosphate linkage.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a chirally pure mipomersen composition. That is to say,in some embodiments, a provided chirally controlled oligonucleotidecomposition provides mipomersen as a single diastereomer with respect tothe configuration of the linkage phosphorus. In some embodiments, aprovided chirally controlled oligonucleotide composition is a chirallyuniform mipomersen composition. That is to say, in some embodiments,every linkage phosphorus of mipomersen is in the Rp configuration orevery linkage phosphorus of mipomersen is in the Sp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of one or more providedoligonucleotide types. One of skill in the chemical and medicinal artswill recognize that the selection and amount of each of the one or moretypes of provided oligonucleotides in a provided composition will dependon the intended use of that composition. That is to say, one of skill inthe relevant arts would design a provided chirally controlledoligonucleotide composition such that the amounts and types of providedoligonucleotides contained therein cause the composition as a whole tohave certain desirable characteristics (e.g., biologically desirable,therapeutically desirable, etc.).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of two or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of threeor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof four or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of five or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of six or more provided oligonucleotide types.In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of seven or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of eightor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof nine or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of ten or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of fifteen or more provided oligonucleotidetypes.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration and an amount of chirally uniform mipomersen ofthe Sp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration, an amount of chirally uniform mipomersen of theSp configuration, and an amount of one or more chirally pure mipomersenof a desired diastereomeric form.

In some embodiments, a provided oligonucleotide type is selected fromthose described in PCT/US2013/050407, which is incorporated herein byreference. In some embodiments, a provided chirally controlledoligonucleotide composition comprises oligonucleotides of anoligonucleotide type selected from those described in PCT/US2013/050407.

Example Methods for Preparing Oligonucleotides and Compositions

The present disclosure provides methods for making chirally controlledoligonucleotides and chirally controlled compositions comprising one ormore specific nucleotide types. In some embodiments, the phrase“oligonucleotide type,” as used herein, defines an oligonucleotide thathas a particular base sequence, pattern of backbone linkages, pattern ofbackbone chiral centers, and pattern of backbone phosphorusmodifications (e.g., “—XLR¹” groups). Oligonucleotides of a commondesignated “type” are structurally identical to one another with respectto base sequence, pattern of backbone linkages, pattern of backbonechiral centers, and pattern of backbone phosphorus modifications. Insome embodiments, oligonucleotides of an oligonucleotide type areidentical.

In some embodiments, a provided chirally controlled oligonucleotide inthe disclosure has properties different from those of the correspondingstereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has lipophilicity different from that of thestereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has different retention time on HPLC. In someembodiments, a chirally controlled oligonucleotide may have a peakretention time significantly different from that of the correspondingstereorandom oligonucleotide mixture. During oligonucleotidepurification using HPLC as generally practiced in the art, certainchirally controlled oligonucleotides will be largely if not totallylost. During oligonucleotide purification using HPLC as generallypracticed in the art, certain chirally controlled oligonucleotides willbe largely if not totally lost. One of the consequences is that certaindiastereomers of a stereorandom oligonucleotide mixture (certainchirally controlled oligonucleotides) are not tested in assays. Anotherconsequence is that from batches to batches, due to the inevitableinstrumental and human errors, the supposedly “pure” stereorandomoligonucleotide will have inconsistent compositions in thatdiastereomers in the composition, and their relative and absoluteamounts, are different from batches to batches. The chirally controlledoligonucleotide and chirally controlled oligonucleotide compositionprovided in this disclosure overcome such problems, as a chirallycontrolled oligonucleotide is synthesized in a chirally controlledfashion as a single diastereomer, and a chirally controlledoligonucleotide composition comprise predetermined levels of one or moreindividual oligonucleotide types.

One of skill in the chemical and synthetic arts will appreciate thatsynthetic methods of the present disclosure provide for a degree ofcontrol during each step of the synthesis of a provided oligonucleotidesuch that each nucleotide unit of the oligonucleotide can be designedand/or selected in advance to have a particular stereochemistry at thelinkage phosphorus and/or a particular modification at the linkagephosphorus, and/or a particular base, and/or a particular sugar. In someembodiments, a provided oligonucleotide is designed and/or selected inadvance to have a particular combination of stereocenters at the linkagephosphorus of the internucleotidic linkage.

In some embodiments, a provided oligonucleotide made using methods ofthe present disclosure is designed and/or determined to have aparticular combination of linkage phosphorus modifications. In someembodiments, a provided oligonucleotide made using methods of thepresent disclosure is designed and/or determined to have a particularcombination of bases. In some embodiments, a provided oligonucleotidemade using methods of the present disclosure is designed and/ordetermined to have a particular combination of sugars. In someembodiments, a provided oligonucleotide made using methods of thepresent disclosure is designed and/or determined to have a particularcombination of one or more of the above structural characteristics.

Methods of the present disclosure exhibit a high degree of chiralcontrol. For instance, methods of the present disclosure facilitatecontrol of the stereochemical configuration of every single linkagephosphorus within a provided oligonucleotide. In some embodiments,methods of the present disclosure provide an oligonucleotide comprisingone or more modified internucleotidic linkages independently having thestructure of formula I.

In some embodiments, methods of the present disclosure provide anoligonucleotide which is a mipomersen unimer. In some embodiments,methods of the present disclosure provide an oligonucleotide which is amipomersen unimer of configuration Rp. In some embodiments, methods ofthe present disclosure provide an oligonucleotide which is a mipomersenunimer of configuration Sp.

In some embodiments, methods of the present disclosure provide achirally controlled oligonucleotide composition, i.e., anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. In some embodiments a chirallycontrolled oligonucleotide composition comprises one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises more than one oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide composition comprisesa plurality of oligonucleotide types. Example chirally controlledoligonucleotide compositions made in accordance with the presentdisclosure are described herein.

In some embodiments, methods of the present disclosure provide chirallypure mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent disclosure provide compositions of mipomersen wherein mipomersenexists in the composition in the form of a single diastereomer withrespect to the configuration of the linkage phosphorus.

In some embodiments, methods of the present disclosure provide chirallyuniform mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent disclosure provide compositions of mipomersen in which allnucleotide units therein have the same stereochemistry with respect tothe configuration of the linkage phosphorus, e.g., all nucleotide unitsare of the Rp configuration at the linkage phosphorus or all nucleotideunits are of the Sp configuration at the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotide isover 50% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 55% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 60% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 65% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 70% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 75% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 80% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 85% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 90% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 91% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 92% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 93% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 94% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 95% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 96% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 97% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 98% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 99% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 99.5% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 99.6% pure. In some embodiments, aprovided chirally controlled oligonucleotide is over about 99.7% pure.In some embodiments, a provided chirally controlled oligonucleotide isover about 99.8% pure. In some embodiments, a provided chirallycontrolled oligonucleotide is over about 99.9% pure. In someembodiments, a provided chirally controlled oligonucleotide is over atleast about 99% pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise a single oligonucleotide type. Incertain embodiments, such compositions are about 50% diastereomericallypure. In some embodiments, such compositions are about 50%diastereomerically pure. In some embodiments, such compositions areabout 50% diastereomerically pure. In some embodiments, suchcompositions are about 55% diastereomerically pure. In some embodiments,such compositions are about 60% diastereomerically pure. In someembodiments, such compositions are about 65% diastereomerically pure. Insome embodiments, such compositions are about 70% diastereomericallypure. In some embodiments, such compositions are about 75%diastereomerically pure. In some embodiments, such compositions areabout 80% diastereomerically pure. In some embodiments, suchcompositions are about 85% diastereomerically pure. In some embodiments,such compositions are about 90% diastereomerically pure. In someembodiments, such compositions are about 91% diastereomerically pure. Insome embodiments, such compositions are about 92% diastereomericallypure. In some embodiments, such compositions are about 93%diastereomerically pure. In some embodiments, such compositions areabout 94% diastereomerically pure. In some embodiments, suchcompositions are about 95% diastereomerically pure. In some embodiments,such compositions are about 96% diastereomerically pure. In someembodiments, such compositions are about 97% diastereomerically pure. Insome embodiments, such compositions are about 98% diastereomericallypure. In some embodiments, such compositions are about 99%diastereomerically pure. In some embodiments, such compositions areabout 99.5% diastereomerically pure. In some embodiments, suchcompositions are about 99.6% diastereomerically pure. In someembodiments, such compositions are about 99.7% diastereomerically pure.In some embodiments, such compositions are about 99.8%diastereomerically pure. In some embodiments, such compositions areabout 99.9% diastereomerically pure. In some embodiments, suchcompositions are at least about 99% diastereomerically pure.

Among other things, the present disclosure recognizes the challenge ofstereoselective (rather than stereorandom or racemic) preparation ofoligonucleotides. Among other things, the present disclosure providesmethods and reagents for stereoselective preparation of oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10)internucleotidic linkages, and particularly for oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiralinternucleotidic linkages. In some embodiments, in a stereorandom orracemic preparation of oligonucleotides, at least one chiralinternucleotidic linkage is formed with less than 90:10, 95:5, 96:4,97:3, or 98:2 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 90:10,95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, fora stereoselective or chirally controlled preparation ofoligonucleotides, each chiral internucleotidic linkage is formed withgreater than 95:5 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 96:4diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 97:3diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 98:2diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 99:1diastereoselectivity. In some embodiments, diastereoselectivity of achiral internucleotidic linkage in an oligonucleotide may be measuredthrough a model reaction, e.g. formation of a dimer under essentiallythe same or comparable conditions wherein the dimer has the sameinternucleotidic linkage as the chiral internucleotidic linkage, the5′-nucleoside of the dimer is the same as the nucleoside to the 5′-endof the chiral internucleotidic linkage, and the 3′-nucleoside of thedimer is the same as the nucleoside to the 3′-end of the chiralinternucleotidic linkage.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise multiple oligonucleotide types. Insome embodiments, methods of the present disclosure allow for thegeneration of a library of chirally controlled oligonucleotides suchthat a pre-selected amount of any one or more chirally controlledoligonucleotide types can be mixed with any one or more other chirallycontrolled oligonucleotide types to create a chirally controlledoligonucleotide composition. In some embodiments, the pre-selectedamount of an oligonucleotide type is a composition having any one of theabove-described diastereomeric purities.

In some embodiments, the present disclosure provides methods for makinga chirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved.

When describing the provided methods, the word “cycle” has its ordinarymeaning as understood by a person of ordinary skill in the art. In someembodiments, one round of steps (1)-(4) is referred to as a cycle.

In some embodiments, the present disclosure provides methods for makingchirally controlled oligonucleotide compositions, comprising steps of:

(a) providing an amount of a first chirally controlled oligonucleotide;and

(b) optionally providing an amount of one or more additional chirallycontrolled oligonucleotides.

In some embodiments, a first chirally controlled oligonucleotide is anoligonucleotide type, as described herein. In some embodiments, a one ormore additional chirally controlled oligonucleotide is a one or moreoligonucleotide type, as described herein.

One of skill in the relevant chemical and synthetic arts will recognizethe degree of versatility and control over structural variation andstereochemical configuration of a provided oligonucleotide whensynthesized using methods of the present disclosure. For instance, aftera first cycle is complete, a subsequent cycle can be performed using anucleotide unit individually selected for that subsequent cycle which,in some embodiments, comprises a nucleobase and/or a sugar that isdifferent from the first cycle nucleobase and/or sugar. Likewise, thechiral auxiliary used in the coupling step of the subsequent cycle canbe different from the chiral auxiliary used in the first cycle, suchthat the second cycle generates a phosphorus linkage of a differentstereochemical configuration. In some embodiments, the stereochemistryof the linkage phosphorus in the newly formed internucleotidic linkageis controlled by using stereochemically pure phosphoramidites.Additionally, the modification reagent used in the modifying step of asubsequent cycle can be different from the modification reagent used inthe first or former cycle. The cumulative effect of this iterativeassembly approach is such that each component of a providedoligonucleotide can be structurally and configurationally tailored to ahigh degree. An additional advantage to this approach is that the stepof capping minimizes the formation of “n−1” impurities that wouldotherwise make isolation of a provided oligonucleotide extremelychallenging, and especially oligonucleotides of longer lengths.

In some embodiments, an example cycle of the method for making chirallycontrolled oligonucleotides is illustrated in example schemes describedin the present disclosure. In some embodiments, an example cycle of themethod for making chirally controlled oligonucleotides is illustrated inScheme I. In some embodiments,

represents the solid support, and optionally a portion of the growingchirally controlled oligonucleotide attached to the solid support. Thechiral auxiliary exemplified has the structure of formula 3-I:

which is further described below. “Cap” is any chemical moietyintroduced to the nitrogen atom by the capping step, and in someembodiments, is an amino protecting group. One of ordinary skill in theart understands that in the first cycle, there may be only onenucleoside attached to the solid support when started, and cycle exitcan be performed optionally before deblocking. As understood by a personof skill in the art, B^(PRO) is a protected base used in oligonucleotidesynthesis. Each step of the above-depicted cycle of Scheme I isdescribed further below.

Synthesis on Solid Support

In some embodiments, the synthesis of a provided oligonucleotide isperformed on solid phase. In some embodiments, reactive groups presenton a solid support are protected. In some embodiments, reactive groupspresent on a solid support are unprotected. During oligonucleotidesynthesis a solid support is treated with various reagents in severalsynthesis cycles to achieve the stepwise elongation of a growingoligonucleotide chain with individual nucleotide units. The nucleosideunit at the end of the chain which is directly linked to the solidsupport is termed “the first nucleoside” as used herein. A firstnucleoside is bound to a solid support via a linker moiety, i.e. adiradical with covalent bonds between either of a CPG, a polymer orother solid support and a nucleoside. The linker stays intact during thesynthesis cycles performed to assemble the oligonucleotide chain and iscleaved after the chain assembly to liberate the oligonucleotide fromthe support.

Solid supports for solid-phase nucleic acid synthesis include thesupports described in, e.g., U.S. Pat. Nos. 4,659,774, 5,141,813,4,458,066; Caruthers U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, and 5,132,418; Andrus et al. U.S. Pat. Nos.5,047,524, 5,262,530; and Koster U.S. Pat. No. 4,725,677 (reissued asRE34,069). In some embodiments, a solid phase is an organic polymersupport. In some embodiments, a solid phase is an inorganic polymersupport. In some embodiments, an organic polymer support is polystyrene,aminomethyl polystyrene, a polyethylene glycol-polystyrene graftcopolymer, polyacrylamide, polymethacrylate, polyvinylalcohol, highlycross-linked polymer (HCP), or other synthetic polymers, carbohydratessuch as cellulose and starch or other polymeric carbohydrates, or otherorganic polymers and any copolymers, composite materials or combinationof the above inorganic or organic materials. In some embodiments, aninorganic polymer support is silica, alumina, controlled polyglass(CPG), which is a silica-gel support, or aminopropyl CPG. Other usefulsolid supports include fluorous solid supports (see e.g.,WO/2005/070859), long chain alkylamine (LCAA) controlled pore glass(CPG) solid supports (see e.g., S. P. Adams, K. S. Kavka, E. J. Wykes,S. B. Holder and G. R. Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663;G. R. Gough, M. J. Bruden and P. T. Gilham, Tetrahedron Lett., 1981, 22,4177-4180). Membrane supports and polymeric membranes (see e.g.Innovation and Perspectives in Solid Phase Synthesis, Peptides, Proteinsand Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and U.S. Pat.No. 4,923,901) are also useful for the synthesis of nucleic acids. Onceformed, a membrane can be chemically functionalized for use in nucleicacid synthesis. In addition to the attachment of a functional group tothe membrane, the use of a linker or spacer group attached to themembrane is also used in some embodiments to minimize steric hindrancebetween the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the artto be suitable for use in solid phase methodologies, including, forexample, glass sold as Primer™ 200 support, controlled pore glass (CPG),oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic AcidsResearch, 1991, 19, 1527), TentaGel Support—an aminopolyethyleneglycolderivatized support (see, e.g., Wright, et al., Tetrahedron Lett., 1993,34, 3373), and Poros-a copolymer of polystyrene/divinylbenzene.

Surface activated polymers have been demonstrated for use in synthesisof natural and modified nucleic acids and proteins on several solidsupports mediums. A solid support material can be any polymer suitablyuniform in porosity, having sufficient amine content, and sufficientflexibility to undergo any attendant manipulations without losingintegrity. Examples of suitable selected materials include nylon,polypropylene, polyester, polytetrafluoroethylene, polystyrene,polycarbonate, and nitrocellulose. Other materials can serve as a solidsupport, depending on the design of the investigator. In considerationof some designs, for example, a coated metal, in particular gold orplatinum can be selected (see e.g., US publication No. 20010055761). Inone embodiment of oligonucleotide synthesis, for example, a nucleosideis anchored to a solid support which is functionalized with hydroxyl oramino residues. Alternatively, a solid support is derivatized to providean acid labile trialkoxytrityl group, such as a trimethoxytrityl group(TMT). Without being bound by theory, it is expected that the presenceof a trialkoxytrityl protecting group will permit initial detritylationunder conditions commonly used on DNA synthesizers. For a faster releaseof oligonucleotide material in solution with aqueous ammonia, adiglycoate linker is optionally introduced onto the support.

In some embodiments, a provided oligonucleotide alternatively issynthesized from the 5′ to 3′ direction. In some embodiments, a nucleicacid is attached to a solid support through its 5′ end of the growingnucleic acid, thereby presenting its 3′ group for reaction, i.e. using5′-nucleoside phosphoramidites or in enzymatic reaction (e.g. ligationand polymerization using nucleoside 5′-triphosphates). When consideringthe 5′ to 3′ synthesis the iterative steps of the present disclosureremain unchanged (i.e. capping and modification on the chiralphosphorus).

Linking Moiety

A linking moiety or linker is optionally used to connect a solid supportto a compound comprising a free nucleophilic moiety. Suitable linkersare known such as short molecules which serve to connect a solid supportto functional groups (e.g., hydroxyl groups) of initial nucleosidesmolecules in solid phase synthetic techniques. In some embodiments, thelinking moiety is a succinamic acid linker, or a succinate linker(—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). In some embodiments,the linking moiety and the nucleoside are bonded together through anester bond. In some embodiments, a linking moiety and a nucleoside arebonded together through an amide bond. In some embodiments, a linkingmoiety connects a nucleoside to another nucleotide or nucleic acid.Suitable linkers are disclosed in, for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon,R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28.

A linker moiety is used to connect a compound comprising a freenucleophilic moiety to another nucleoside, nucleotide, or nucleic acid.In some embodiments, a linking moiety is a phosphodiester linkage. Insome embodiments, a linking moiety is an H-phosphonate moiety. In someembodiments, a linking moiety is a modified phosphorus linkage asdescribed herein. In some embodiments, a universal linker (UnyLinker) isused to attached the oligonucleotide to the solid support (Ravikumar etal., Org. Process Res. Dev., 2008, 12 (3), 399-410). In someembodiments, other universal linkers are used (Pon, R. T., Curr. Prot.Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, variousorthogonal linkers (such as disulfide linkers) are used (Pon, R. T.,Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

Among other things, the present disclosure recognizes that a linker canbe chosen or designed to be compatible with a set of reaction conditionsemployed in oligonucleotide synthesis. In some embodiments, to avoiddegradation of oligonucleotides and to avoid desulfurization, auxiliarygroups are selectively removed before de-protection. In someembodiments, DPSE group can selectively be removed by F⁻ ions. In someembodiments, the present disclosure provides linkers that are stableunder a DPSE de-protection condition, e.g., 0.1M TBAF in MeCN, 0.5MHF-Et₃N in THF or MeCN, etc. In some embodiments, a provided linker isthe SP linker. In some embodiments, the present disclosure demonstratesthat the SP linker is stable under a DPSE de-protection condition, e.g.,0.1M TBAF in MeCN, 0.5M HF-Et₃N in THF or MeCN, etc.; they are alsostable, e.g., under anhydrous basic conditions, such as om1M DBU inMeCN.

In some embodiments, an example linker is:

In some embodiments, the succinyl linker, Q-linker or oxalyl linker isnot stable to one or more DPSE-deprotection conditions using F⁻.

General Conditions—Solvents for Synthesis

Syntheses of provided oligonucleotides are generally performed inaprotic organic solvents. In some embodiments, a solvent is a nitrilesolvent such as, e.g., acetonitrile. In some embodiments, a solvent is abasic amine solvent such as, e.g., pyridine. In some embodiments, asolvent is an ethereal solvent such as, e.g., tetrahydrofuran. In someembodiments, a solvent is a halogenated hydrocarbon such as, e.g.,dichloromethane. In some embodiments, a mixture of solvents is used. Incertain embodiments a solvent is a mixture of any one or more of theabove-described classes of solvents.

In some embodiments, when an aprotic organic solvent is not basic, abase is present in the reacting step. In some embodiments where a baseis present, the base is an amine base such as, e.g., pyridine,quinoline, or N,N-dimethylaniline. Examples of other amine bases includepyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline,N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

In some embodiments, a base is other than an amine base.

In some embodiments, an aprotic organic solvent is anhydrous. In someembodiments, an anhydrous aprotic organic solvent is freshly distilled.In some embodiments, a freshly distilled anhydrous aprotic organicsolvent is a basic amine solvent such as, e.g., pyridine. In someembodiments, a freshly distilled anhydrous aprotic organic solvent is anethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, afreshly distilled anhydrous aprotic organic solvent is a nitrile solventsuch as, e.g., acetonitrile.

Chiral Reagent/Chiral Auxiliary

In some embodiments, chiral reagents are used to conferstereoselectivity in the production of chirally controlledolignucleotides. Many different chiral reagents, also referred to bythose of skill in the art and herein as chiral auxiliaries, may be usedin accordance with methods of the present disclosure. Examples of suchchiral reagents are described herein and in Wada I, II and III,referenced above. In certain embodiments, a chiral reagent is asdescribed by Wada I. In some embodiments, a chiral reagent for use inaccordance with the methods of the present disclosure are of Formula3-I, below:

wherein W¹ and W² are any of —O—, —S—, or —NG⁵-, U₁ and U₃ are carbonatoms which are bonded to U₂ if present, or to each other if r is 0, viaa single, double or triple bond. U₂ is —C—, —CG⁸-, —CG⁸G⁸-, —NG⁸-, —N—,—O—, or —S— where r is an integer of 0 to 5 and no more than twoheteroatoms are adjacent. When any one of U₂ is C, a triple bond must beformed between a second instance of U₂, which is C, or to one of U₁ orU₃. Similarly, when any one of U₂ is CG⁸, a double bond is formedbetween a second instance of U₂ which is —CG⁸- or —N—, or to one of U₁or U₃.

In some embodiments, —U₁(G₃G₄)-(U₂)_(r)—U₃(G₁G₂)- is -CG³G⁴-CG¹G²-. Insome embodiments, —U₁—(U₂)_(r)—U₃— is —CG³=CG¹-. In some embodiments,—U₁—(U₂)_(r)—U₃— is —C≡C—. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³=CG-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-O-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-NG-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is—CG³G⁴-N-CG²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is —CG³G⁴-N═CG⁸-CG¹G²-.

As defined herein, G¹, G², G³, G⁴, G⁵, and G⁸ are independentlyhydrogen, or an optionally substituted group selected from alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, andaryl; or two of G¹, G², G³, G⁴, and G⁵ are G⁶ (taken together to form anoptionally substituted, saturated, partially unsaturated or unsaturatedcarbocyclic or heteroatom-containing ring of up to about 20 ring atomswhich is monocyclic or polycyclic, and is fused or unfused). In someembodiments, a ring so formed is substituted by oxo, thioxo, alkyl,alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments,when a ring formed by taking two G⁶ together is substituted, it issubstituted by a moiety which is bulky enough to conferstereoselectivity during the reaction.

In some embodiments, a ring formed by taking two of G⁶ together isoptionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl,cyclopentenyl, tetrahydropyranyl, or piperazinyl. In some embodiments, aring formed by taking two of G⁶ together is optionally substitutedcyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl,tetrahydropyranyl, pyrrolidinyl, or piperazinyl.

In some embodiments, G¹ is optionally substituted phenyl. In someembodiments, G¹ is phenyl. In some embodiments, G² is methyl orhydrogen. In some embodiments, G¹ is optionally substituted phenyl andG² is methyl. In some embodiments, G¹ is phenyl and G² is methyl.

In some embodiments, r is 0.

In some embodiments, W¹ is —NG⁵-. In some embodiments, one of G³ and G⁴is taken together with G⁵ to form an optionally substituted pyrrolidinylring. In some embodiments, one of G³ and G⁴ is taken together with G⁵ toform a pyrrolidinyl ring.

In some embodiments, W² is —O—.

In some embodiments, a chiral reagent is a compound of Formula 3-AA:

wherein each variable is independently as defined above and describedherein.

In some embodiments of Formula 3AA, W¹ and W² are independently —NG⁵-,—O—, or —S—; G¹, G², G³, G⁴, and G⁵ are independently hydrogen, or anoptionally substituted group selected from alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl; or two of G¹, G²,G³, G⁴, and G⁵ are G⁶ (taken together to form an optionally substitutedsaturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused), and no more than four ofG¹, G², G³, G⁴, and G⁵ are G⁶. Similarly to the compounds of Formula3-I, any of G¹, G², G³, G⁴, or G⁵ are optionally substituted by oxo,thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In someembodiments, such substitution induces stereoselectivity in chirallycontrolled oligonucleotide production.

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, W¹ is —NG⁵, W² is O, each of G¹ and G³ isindependently hydrogen or an optionally substituted group selected fromC₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl, G² is —C(R)₂Si(R)₃,and G⁴ and G⁵ are taken together to form an optionally substitutedsaturated, partially unsaturated or unsaturated heteroatom-containingring of up to about 20 ring atoms which is monocyclic or polycyclic,fused or unfused. In some embodiments, each R is independently hydrogen,or an optionally substituted group selected from C₁-C₆ aliphatic,carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, G²is —C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, andeach R of —Si(R)₃ is independently an optionally substituted groupselected from C₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl. Insome embodiments, at least one R of —Si(R)₃ is independently optionallysubstituted C₁₋₁₀ alkyl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted phenyl. In some embodiments, oneR of —Si(R)₃ is independently optionally substituted phenyl, and each ofthe other two R is independently optionally substituted C₁₋₁₀ alkyl. Insome embodiments, one R of —Si(R)₃ is independently optionallysubstituted C₁₋₁₀ alkyl, and each of the other two R is independentlyoptionally substituted phenyl. In some embodiments, G² is optionallysubstituted —CH₂Si(Ph)(Me)₂. In some embodiments, G² is optionallysubstituted —CH₂Si(Me)(Ph)₂. In some embodiments, G² is —CH₂Si(Me)(Ph)₂.In some embodiments, G⁴ and G⁵ are taken together to form an optionallysubstituted saturated 5-6 membered ring containing one nitrogen atom (towhich G⁵ is attached). In some embodiments, G⁴ and G⁵ are taken togetherto form an optionally substituted saturated 5-membered ring containingone nitrogen atom. In some embodiments, G¹ is hydrogen. In someembodiments, G³ is hydrogen. In some embodiments, both G¹ and G³ arehydrogen.

In some embodiments, a chiral reagent has one of the following formulae:

In some embodiments, a chiral reagent is an aminoalcohol. In someembodiments, a chiral reagent is an aminothiol. In some embodiments, achiral reagent is an aminophenol. In some embodiments, a chiral reagentis (S)- and (R)-2-methylamino-1-phenylethanol, (1R,2S)-ephedrine, or(1R,2S)-2-methylamino-1,2-diphenylethanol.

In some embodiments of the disclosure, a chiral reagent is a compound ofone of the following formulae:

As demonstrated herein, when used for preparing a chiralinternucleotidic linkage, to obtain stereoselectivity generallystereochemically pure chiral reagents are utilized. Among other things,the present disclosure provides stereochemically pure chiral reagents,including those having structures described.

The choice of chiral reagent, for example, the isomer represented byFormula Q or its stereoisomer, Formula R, permits specific control ofchirality at a linkage phosphorus. Thus, either an Rp or Spconfiguration can be selected in each synthetic cycle, permittingcontrol of the overall three dimensional structure of a chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide has all Rp stereocenters. In some embodiments of thedisclosure, a chirally controlled oligonucleotide has all Spstereocenters. In some embodiments of the disclosure, each linkagephosphorus in the chirally controlled oligonucleotide is independentlyRp or Sp. In some embodiments of the disclosure, each linkage phosphorusin the chirally controlled oligonucleotide is independently Rp or Sp,and at least one is Rp and at least one is Sp. In some embodiments, theselection of Rp and Sp centers is made to confer a specific threedimensional superstructure to a chirally controlled oligonucleotide.Examples of such selections are described in further detail herein.

In some embodiments, a chiral reagent for use in accordance with thepresent disclosure is selected for its ability to be removed at aparticular step in the above-depicted cycle. For example, in someembodiments it is desirable to remove a chiral reagent during the stepof modifying the linkage phosphorus. In some embodiments, it isdesirable to remove a chiral reagent before the step of modifying thelinkage phosphorus. In some embodiments, it is desirable to remove achiral reagent after the step of modifying the linkage phosphorus. Insome embodiments, it is desirable to remove a chiral reagent after afirst coupling step has occurred but before a second coupling step hasoccurred, such that a chiral reagent is not present on the growingoligonucleotide during the second coupling (and likewise for additionalsubsequent coupling steps). In some embodiments, a chiral reagent isremoved during the “deblock” reaction that occurs after modification ofthe linkage phosphorus but before a subsequent cycle begins. Examplemethods and reagents for removal are described herein.

In some embodiments, removal of chiral auxiliary is achieved whenperforming the modification and/or deblocking step, as illustrated inScheme I. It can be beneficial to combine chiral auxiliary removaltogether with other transformations, such as modification anddeblocking. A person of ordinary skill in the art would appreciate thatthe saved steps/transformation could improve the overall efficiency ofsynthesis, for instance, with respect to yield and product purity,especially for longer oligonucleotides. One example wherein the chiralauxiliary is removed during modification and/or deblocking isillustrated in Scheme I.

In some embodiments, a chiral reagent for use in accordance with methodsof the present disclosure is characterized in that it is removable undercertain conditions. For instance, in some embodiments, a chiral reagentis selected for its ability to be removed under acidic conditions. Incertain embodiments, a chiral reagent is selected for its ability to beremoved under mildly acidic conditions. In certain embodiments, a chiralreagent is selected for its ability to be removed by way of an E1elimination reaction (e.g., removal occurs due to the formation of acation intermediate on the chiral reagent under acidic conditions,causing the chiral reagent to cleave from the oligonucleotide). In someembodiments, a chiral reagent is characterized in that it has astructure recognized as being able to accommodate or facilitate an E1elimination reaction. One of skill in the relevant arts will appreciatewhich structures would be envisaged as being prone toward undergoingsuch elimination reactions.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile. In some embodiments, a chiral reagent isselected for its ability to be removed with an amine nucleophile. Insome embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile other than an amine.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a base. In some embodiments, a chiral reagent is selectedfor its ability to be removed with an amine. In some embodiments, achiral reagent is selected for its ability to be removed with a baseother than an amine.

Additional chiral auxiliaries and their use can be found in e.g., Wada I(JP4348077; WO2005/014609; WO2005/092909), Wada II (WO2010/064146), WadaIII (WO2012/039448), Chiral Control (WO2010/064146), etc.

Activation

An achiral H-phosphonate moiety is treated with the first activatingreagent to form the first intermediate. In one embodiment, the firstactivating reagent is added to the reaction mixture during thecondensation step. Use of the first activating reagent is dependent onreaction conditions such as solvents that are used for the reaction.Examples of the first activating reagent are phosgene, trichloromethylchloroformate, bis(trichloromethyl)carbonate (BTC), oxalyl chloride,Ph₃PCl₂, (PhO)₃PCl₂, N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP).

The example of achiral H-phosphonate moiety is a compound shown in theabove Scheme. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene. H⁺DBUmay be, for example, ammonium ion, alkylammonium ion, heteroaromaticiminium ion, or heterocyclic iminium ion, any of which is primary,secondary, tertiary or quaternary, or a monovalent metal ion.

Reacting with Chiral Reagent

After the first activation step, the activated achiral H-phosphonatemoiety reacts with a chiral reagent, which is represented by formula(Z-I) or (Z-I′), to form a chiral intermediate of formula (Z-Va),(Z-Vb), (Z-Va′), or (Z-Vb′).

Stereospecific Condensation Step

A chiral intermediate of Formula Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) istreated with the second activating reagent and a nucleoside to form acondensed intermediate. The nucleoside may be on solid support. Examplesof the second activating reagent are 4,5-dicyanoimidazole (DCI),4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole(NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole(BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate(CMPT), N-cyanomethylpiperidinium triflate,N-cyanomethyldimethylammonium triflate. A chiral intermediate of FormulaZ-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) may be isolated as a monomer.Usually, the chiral intermediate of Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′))is not isolated and undergoes a reaction in the same pot with anucleoside or modified nucleoside to provide a chiral phosphitecompound, a condensed intermediate. In other embodiments, when themethod is performed via solid phase synthesis, the solid supportcomprising the compound is filtered away from side products, impurities,and/or reagents.

Capping Step

If the final nucleic acid is larger than a dimer, the unreacted —OHmoiety is capped with a blocking group and the chiral auxiliary in thecompound may also be capped with a blocking group to form a cappedcondensed intermediate. If the final nucleic acid is a dimer, then thecapping step is not necessary.

Modifying Step

The compound is modified by reaction with an electrophile. The cappedcondensed intermediate may be executed modifying step. In someembodiments, the modifying step is performed using a sulfurelectrophile, a selenium electrophile or a boronating agent. Examples ofmodifying steps are step of oxidation and sulfurization.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:

Z^(z1)—S—S—Z^(z2), or Z^(z1)—S—V^(z)—Z^(z2);  S₈ (Formula Z-B),

wherein Z^(z1) and Z^(z2) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z1) and Z^(z2) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl,alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof following Formulae Z-A, Z-B, Z-C, Z-D, Z-E, or Z-F:

In some embodiments, a sulfurization reagent is3-phenyl-1,2,4-dithiazolin-5-one.

In some embodiments, the selenium electrophile is a compound having oneof the following formulae:

Z^(z3)—Se—Se—Z^(z4), or Z^(z3)—Se—V^(z)—Z^(z4);  Se (Formula Z-G),

wherein Z^(z3) and Z^(z4) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z3) and Z^(z4) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, S, O, or NR^(f); and R^(f) is hydrogen,alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, the selenium electrophile is a compound of FormulaZ-G, Z-H, Z-I, Z-J, Z-K, or Z-L.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃DIPEA), borane-pyridine (BH₃Py),borane-2-chloropyridine (BH₃CPy), borane-aniline (BH₃An),borane-tetrahydrofurane (BH₃THF), or borane-dimethylsulfide (BH₃Me₂S).

In some embodiments, after the modifying step, a chiral auxiliary groupfalls off from the growing oligonucleotide chain. In some embodiments,after the modifying step, a chiral auxiliary group remains connected tothe internucleotidic phosphorus atom.

In some embodiments of the method, the modifying step is an oxidationstep. In some embodiments of the method, the modifying step is anoxidation step using similar conditions as described above in thisapplication. In some embodiments, an oxidation step is as disclosed in,e.g., JP 2010-265304 A and WO2010/064146.

Chain Elongation Cycle and De-Protection Step

The capped condensed intermediate is deblocked to remove the blockinggroup at the 5′-end of the growing nucleic acid chain to provide acompound. The compound is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate, a capped condensedintermediate, a modified capped condensed intermediate, and a5′-deprotected modified capped intermediate. Following at least oneround of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups for, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid. In other embodiments, the nucleoside comprising a 5′-OHmoiety is an intermediate from a previous chain elongation cycle asdescribed herein. In yet other embodiments, the nucleoside comprising a5′-OH moiety is an intermediate obtained from another known nucleic acidsynthetic method. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification.

In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic method.In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic methodas described in this application. In yet other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate obtained fromanother known nucleic acid synthetic method comprising one or morecycles illustrated in Scheme I. In yet other embodiments, the nucleosidecomprising a 5′-OH moiety is an intermediate obtained from another knownnucleic acid synthetic method comprising one or more cycles illustratedin Scheme I-b, I-c or I-d.

In some embodiments, the present disclosure provides oligonucleotidesynthesis methods that use stable and commercially available materialsas starting materials. In some embodiments, the present disclosureprovides oligonucleotide synthesis methods to produce stereocontrolledphosphorus atom-modified oligonucleotide derivatives using an achiralstarting material.

In some embodiments, the method of the present disclosure does not causedegradations under the de-protection steps. Further the method does notrequire special capping agents to produce phosphorus atom-modifiedoligonucleotide derivatives.

Condensing Reagent

Condensing reagents (C_(R)) useful in accordance with methods of thepresent disclosure are of any one of the following general formulae:

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, and Z⁹ are independentlyoptionally substituted group selected from alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸ and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ and Z⁹are taken together to form a 3 to 20 membered alicyclic or heterocyclicring; Q⁻ is a counter anion; and LG is a leaving group.

In some embodiments, a counter ion of a condensing reagent C_(R) is Cl⁻,Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf isCF₃SO₂. In some embodiments, a leaving group of a condensing reagentC_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

Examples of condensing reagents used in accordance with methods of thepresent disclosure include, but are not limited to, pentafluorobenzoylchloride, carbonyldiimidazole (CDI),1-mesitylenesulfonyl-3-nitrotriazole (MSNT),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl),benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(PyBOP), N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),DIPCDI; N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic bromide (BopBr),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bromotripyrrolidinophosphoniumhexafluorophosphate (PyBrOP);O—(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU); and tetramethylfluoroformamidinium hexafluorophosphate (TFFH).In certain embodiments, a counter ion of the condensing reagent C_(R) isCl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, whereinTf is CF₃SO₂.

In some embodiments, a condensing reagent is 1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl) tetrazolide, pivaloylchloride, bromotrispyrrolidinophosphonium hexafluorophosphate,N,N′-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), or2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane. In some embodiment,a condensing reagent is N,N′-bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BopCl). In some embodiments, a condensing reagent is selectedfrom those described in WO/2006/066260).

In some embodiments, a condensing reagent is1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP):

Selection of Base and Sugar of Nucleoside Coupling Partner

As described herein, nucleoside coupling partners for use in accordancewith methods of the present disclosure can be the same as one another orcan be different from one another. In some embodiments, nucleosidecoupling partners for use in the synthesis of a provided oligonucleotideare of the same structure and/or stereochemical configuration as oneanother. In some embodiments, each nucleoside coupling partner for usein the synthesis of a provided oligonucleotide is not of the samestructure and/or stereochemical configuration as certain othernucleoside coupling partners of the oligonucleotide. Example nucleobasesand sugars for use in accordance with methods of the present disclosureare described herein. One of skill in the relevant chemical andsynthetic arts will recognize that any combination of nucleobases andsugars described herein are contemplated for use in accordance withmethods of the present disclosure.

Coupling Step

Example coupling procedures and chiral reagents and condensing reagentsfor use in accordance with the present disclosure are outlined in, interalia, Wada I (JP4348077; WO2005/014609; WO2005/092909), Wada II(WO2010/064146), Wada III (WO2012/039448), and Chiral Control(WO2010/064146). Chiral nucleoside coupling partners for use inaccordance with the present disclosure are also referred to herein as“Wada amidites.” In some embodiments, a coupling partner has thestructure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase, and R¹ is as defined anddescribed herein. In some embodiments, a coupling partner has thestructure of

wherein B^(PRO) is a protected nucleobase, and R¹ is as defined anddescribed herein. In some embodiments, R¹ is optionally substituted C₁₋₆alkyl. In some embodiments, R¹ is Me.

Example chiral phosphoramidites as coupling partner are depicted below:

Additional examples are described in Chiral Control (WO2010/064146).

One of the methods used for synthesizing the coupling partner isdepicted in Scheme II, below.

In some embodiments, the step of coupling comprises reacting a freehydroxyl group of a nucleotide unit of an oligonucleotide with anucleoside coupling partner under suitable conditions to effect thecoupling. In some embodiments, the step of coupling is preceded by astep of deblocking. For instance, in some embodiments, the 5′ hydroxylgroup of the growing oligonucleotide is blocked (i.e., protected) andmust be deblocked in order to subsequently react with a nucleosidecoupling partner.

Once the appropriate hydroxyl group of the growing oligonucleotide hasbeen deblocked, the support is washed and dried in preparation fordelivery of a solution comprising a chiral reagent and a solutioncomprising an activator. In some embodiments, a chiral reagent and anactivator are delivered simultaneously. In some embodiments, co-deliverycomprises delivering an amount of a chiral reagent in solution (e.g., aphosphoramidite solution) and an amount of activator in a solution(e.g., a CMPT solution) in a polar aprotic solvent such as a nitrilesolvent (e.g., acetonitrile).

In some embodiments, the step of coupling provides a crude productcomposition in which the chiral phosphite product is present in adiastereomeric excess of >95%. In some embodiments, the chiral phosphiteproduct is present in a diastereomeric excess of >96%. In someembodiments, the chiral phosphite product is present in a diastereomericexcess of >97%. In some embodiments, the chiral phosphite product ispresent in a diastereomeric excess of >98%. In some embodiments, thechiral phosphite product is present in a diastereomeric excess of >99%.

Capping Step:

Provided methods for making chirally controlled oligonucleotidescomprise a step of capping. In some embodiments, a step of capping is asingle step. In some embodiments, a step of capping is two steps. Insome embodiments, a step of capping is more than two steps.

In some embodiments, a step of capping comprises steps of capping thefree amine of the chiral auxiliary and capping any residual unreacted 5′hydroxyl groups. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with the samecapping group. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with differentcapping groups. In certain embodiments, capping with different cappinggroups allows for selective removal of one capping group over the otherduring synthesis of the oligonucleotide. In some embodiments, thecapping of both groups occurs simultaneously. In some embodiments, thecapping of both groups occurs iteratively.

In certain embodiments, capping occurs iteratively and comprises a firststep of capping the free amine followed by a second step of capping thefree 5′ hydroxyl group, wherein both the free amine and the 5′ hydroxylgroup are capped with the same capping group. For instance, in someembodiments, the free amine of the chiral auxiliary is capped using ananhydride (e.g., phenoxyacetic anhydride, i.e., Pac₂O) prior to cappingof the 5′ hydroxyl group with the same anhydride. In certainembodiments, the capping of the 5′ hydroxyl group with the sameanhydride occurs under different conditions (e.g., in the presence ofone or more additional reagents). In some embodiments, capping of the 5′hydroxyl group occurs in the presence of an amine base in an etherialsolvent (e.g., NMI (N-methylimidazole) in THF). The phrase “cappinggroup” is used interchangeably herein with the phrases “protectinggroup” and “blocking group”.

In some embodiments, an amine capping group is characterized in that iteffectively caps the amine such that it prevents rearrangement and/ordecomposition of the intermediate phosphite species. In someembodiments, a capping group is selected for its ability to protect theamine of the chiral auxiliary in order to prevent intramolecularcleavage of the internucleotide linkage phosphorus.

In some embodiments, a 5′ hydroxyl group capping group is characterizedin that it effectively caps the hydroxyl group such that it prevents theoccurrence of “shortmers,” e.g., “n−m” (m and n are integers and m<n; nis the number of bases in the targeted oligonucleotide) impurities thatoccur from the reaction of an oligonucleotide chain that fails to reactin a first cycle but then reacts in one or more subsequent cycles. Thepresence of such shortmers, especially “n−1”, has a deleterious effectupon the purity of the crude oligonucleotide and makes finalpurification of the oligonucleotide tedious and generally low-yielding.

In some embodiments, a particular cap is selected based on its tendencyto facilitate a particular type of reaction under particular conditions.For instance, in some embodiments, a capping group is selected for itsability to facilitate an E1 elimination reaction, which reaction cleavesthe cap and/or auxiliary from the growing oligonucleotide. In someembodiments, a capping group is selected for its ability to facilitatean E2 elimination reaction, which reaction cleaves the cap and/orauxiliary from the growing oligonucleotide. In some embodiments, acapping group is selected for its ability to facilitate a β-eliminationreaction, which reaction cleaves the cap and/or auxiliary from thegrowing oligonucleotide.

Modifying Step:

As used herein, the phrase “modifying step”, “modification step” and“P-modification step” are used interchangeably and refer generally toany one or more steps used to install a modified internucleotidiclinkage. In some embodiments, the modified internucleotidic linkagehaving the structure of formula I. A P-modification step of the presentdisclosure occurs during assembly of a provided oligonucleotide ratherthan after assembly of a provided oligonucleotide is complete. Thus,each nucleotide unit of a provided oligonucleotide can be individuallymodified at the linkage phosphorus during the cycle within which thenucleotide unit is installed.

In some embodiments, a suitable P-modification reagent is a sulfurelectrophile, selenium electrophile, oxygen electrophile, boronatingreagent, or an azide reagent.

For instance, in some embodiments, a selemium reagent is elementalselenium, a selenium salt, or a substituted diselenide. In someembodiments, an oxygen electrophile is elemental oxygen, peroxide, or asubstituted peroxide. In some embodiments, a boronating reagent is aborane-amine (e.g., N,N-diisopropylethylamine (BH₃.DIPEA),borane-pyridine (BH₃.Py), borane-2-chloropyridine (BH₃.CPy),borane-aniline (BH₃.An)), a borane-ether reagent (e.g.,borane-tetrahydrofuran (BH₃-THF)), a borane-dialkylsulfide reagent(e.g., BH₃.Me₂S), aniline-cyanoborane, or atriphenylphosphine-carboalkoxyborane. In some embodiments, an azidereagent is comprises an azide group capable of undergoing subsequentreduction to provide an amine group.

In some embodiments, a P-modification reagent is a sulfurization reagentas described herein. In some embodiments, a step of modifying comprisessulfurization of phosphorus to provide a phosphorothioate linkage orphosphorothioate triester linkage. In some embodiments, a step ofmodifying provides an oligonucleotide having an internucleotidic linkageof formula I.

In some embodiments, the present disclosure provides sulfurizingreagents, and methods of making, and use of the same.

In some embodiments, such sulfurizing reagents are thiosulfonatereagents. In some embodiments, a thiosulfonate reagent has a structureof formula S-I:

wherein:

R^(s1) is R; and

each of R, L and R¹ is independently as defined and described above andherein.

In some embodiments, the sulfurizing reagent is a bis(thiosulfonate)reagent. In some embodiments, the bis(thiosulfonate) reagent has thestructure of formula S-II:

wherein each of R^(s1) and L is independently as defined and describedabove and herein.

As defined generally above, R^(s1) is R, wherein R is as defined anddescribed above and herein. In some embodiments, R^(s1) is optionallysubstituted aliphatic, aryl, heterocyclyl or heteroaryl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is methyl. In some embodiments, R^(s1) iscyanomethyl. In some embodiments, R^(s1) is nitromethyl. In someembodiments, R^(s1) is optionally substituted aryl. In some embodiments,R^(s1) is optionally substituted phenyl. In some embodiments, R^(s1) isphenyl. In some embodiments, R^(s1) is p-nitrophenyl. In someembodiments, R^(s1) is p-methylphenyl. In some embodiments, R^(s1) isp-chlorophenyl. In some embodiments, R^(s1) is o-chlorophenyl. In someembodiments, R^(s1) is 2,4,6-trichlorophenyl. In some embodiments,R^(s1) is pentafluorophenyl. In some embodiments, R^(s1) is optionallysubstituted heterocyclyl. In some embodiments, R^(s1) is optionallysubstituted heteroaryl.

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments,

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is —S—R^(L3)— or —S—C(O)—R^(L3)—. In some embodiments, Lis —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkenylene. In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—,wherein R^(L3) is an optionally substituted C₁-C₆ alkylene wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkenylene, arylene, or heteroarylene. Insome embodiments, In some embodiments, R^(L3) is an optionallysubstituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆ alkylene)-, —S—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-, —S—CO-arylene-(C₁-C₆ alkylene)-, or—S—CO—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-. In some embodiments,the sulfurizing reagent has the structure of S-I or S-II, wherein L is—S—R^(L3)— or —S—C(O)—R^(L3)—, and the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is alkylene, alkenylene, arylene or heteroarylene.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

In some embodiments, L is

wherein the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is

In some embodiments, R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

wherein the sulfur atom is connected to R¹; and R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is —S—R^(L2), wherein R^(L2) is as defined anddescribed above and herein. In some embodiments, R^(L2) is an optionallysubstituted group selected from —S—(C₁-C₆ alkylene)-heterocyclyl,—S—(C₁-C₆ alkenylene)-heterocyclyl, —S—(C₁-C₆ alkylene)-N(R′)₂,—S—(C₁-C₆ alkylene)-N(R′)₃, wherein each R′ is as defined above anddescribed herein.

In some embodiments, -L-R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

Example bis(thiosulfonate) reagents of formula S—II are depicted below:

In some embodiments, the sulfurization reagent is a compound having oneof the following formulae:

R^(s2)—S—S—R^(s3), or R^(s2)—S—X^(s)—R^(s3),  S₈,

wherein:

-   each of R^(s2) and R^(s3) is independently an optionally substituted    group selected from aliphatic, aminoalkyl, carbocyclyl,    heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,    heteroaryloxy, acyl, amide, imide, or thiocarbonyl; or    -   R^(s2) and R^(s3) are taken together with the atoms to which        they are bound to form an optionally substituted heterocyclic or        heteroaryl ring;-   X^(s) is —S(O)₂—, —O—, or —N(R′)—; and-   R′ is as defined and described above and herein.

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is

Example sulfuring reagents are depicted in Table 5 below.

TABLE 5 Example sulfurization reagents.

S₈

POS

In some embodiments, a provided sulfurization reagent is used to modifyan H-phosphonate. For instance, in some embodiments, an H-phosphonateoligonucleotide is synthesized using, e.g., a method of Wada I or WadaII, and is modified using a sulfurization reagent of formula S-I orS-II:

wherein each of R^(S1), L, and R¹ are as described and defined above andherein.

In some embodiments, the present disclosure provides a process forsynthesizing a phosphorothioate triester, comprising steps of:

i) reacting an H-phosphonate of structure:

wherein each of W, Y, and Z are as described and defined above andherein, with a silylating reagent to provide a silyloxyphosphonate; and

ii) reacting the silyloxyphosphonate with a sulfurization reagent ofstructure S-I or S-II:

to provide a phosphorothiotriester.

In some embodiments, a selenium electrophile is used instead of asulfurizing reagent to introduce modification to the internucleotidiclinkage. In some embodiments, a selenium electrophile is a compoundhaving one of the following formulae:

R^(s2)—Se—Se—R^(s3), or R^(s2)—Se—X^(s)—R^(s3),  Se,

wherein:

-   each of R^(s2) and R^(s3) is independently an optionally substituted    group selected from aliphatic, aminoalkyl, carbocyclyl,    heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy,    heteroaryloxy, acyl, amide, imide, or thiocarbonyl; or    -   R^(s2) and R^(s3) are taken together with the atoms to which        they are bound to form an optionally substituted heterocyclic or        heteroaryl ring;-   X^(s) is —S(O)₂—, —O—, or —N(R′)—; and-   R′ is as defined and described above and herein.

In other embodiments, the selenium electrophile is a compound of Se,KSeCN,

In some embodiments, the selenium electrophile is Se or

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that the moiety transferredto phosphorus during sulfurization is a substituted sulfur (e.g., —SR)as opposed to a single sulfur atom (e.g., —S— or ═S).

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that the activity of thereagent is tunable by modifying the reagent with a certain electronwithdrawing or donating group.

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that it is crystalline. Insome embodiments, a sulfurization reagent for use in accordance with thepresent disclosure is characterized in that it has a high degree ofcrystallinity. In certain embodiments, a sulfurization reagent for usein accordance with the present disclosure is characterized by ease ofpurification of the reagent via, e.g., recrystallization. In certainembodiments, a sulfurization reagent for use in accordance with thepresent disclosure is characterized in that it is substantially freefrom sulfur-containing impurities. In some embodiments, sulfurizationreagents which are substantially free from sulfur-containing impuritiesshow increased efficiency.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages. To synthesize suchchirally controlled oligonucleotides, one or more modifying steps areoptionally replaced with an oxidation step to install the correspondingphosphate diester linkages. In some embodiments, the oxidation step isperformed in a fashion similar to ordinary oligonucleotide synthesis. Insome embodiments, an oxidation step comprises the use of 12. In someembodiments, an oxidation step comprises the use of 12 and pyridine. Insome embodiments, an oxidation step comprises the use of 0.02 M 12 in aTHF/pyridine/water (70:20:10—v/v/v) co-solvent system. An example cycleis depicted in Scheme I-c.

In some embodiments, a phosphorothioate is directly formed throughsulfurization by a sulfurization reagents, e.g.,3-phenyl-1,2,4-dithiazolin-5-one. In some embodiments, after a directinstallation of a phosphorothioate, a chiral auxiliary group remainsattached to the internucleotidic phosphorus atom. In some embodiments,an additional de-protecting step is required to remove the chiralauxiliary (e.g., for DPSE-type chiral auxiliary, using TBAF, HF-Et₃N,etc.).

In some embodiments, a phosphorothioate precursor is used to synthesizechirally controlled oligonucleotides comprising phosphorothioatelinkages. In some embodiments, such a phosphorothioate precursor is

In some embodiments,

is converted into phosphorothioate diester linkages during standarddeprotection/release procedure after cycle exit. Examples are furtherdepicted below.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages and one or morephosphorothioate diester linkages. In some embodiments, the providedchirally controlled oligonucleotide comprises one or more phosphatediester linkages and one or more phosphorothioate diester linkages,wherein at least one phosphate diester linkage is installed after allthe phosphorothioate diester linkages when synthesized from 3′ to 5′. Tosynthesize such chirally controlled oligonucleotides, in someembodiments, one or more modifying steps are optionally replaced with anoxidation step to install the corresponding phosphate diester linkages,and a phosphorothioate precursor is installed for each of thephosphorothioate diester linkages. In some embodiments, aphosphorothioate precursor is converted to a phosphorothioate diesterlinkage after the desired oligonucleotide length is achieved. In someembodiments, the deprotection/release step during or after cycle exitconverts the phosphorothioate precursors into phosphorothioate diesterlinkages. In some embodiments, a phosphorothioate precursor ischaracterized in that it has the ability to be removed by abeta-elimination pathway. In some embodiments, a phosphorothioateprecursor is

As understood by one of ordinary skill in the art, one of the benefitsof using a phosphorothioate precursor, for instance,

during synthesis is that

is more stable than phosphorothioate in certain conditions.

In some embodiments, a phosphorothioate precursor is a phosphorusprotecting group as described herein, e.g., 2-cyanoethyl (CE or Cne),2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl,o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl,3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl,2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl,2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl. Examples are furtherdepicted below.

Methods for synthesizing a desired sulfurization reagent are describedherein and in the examples section.

As noted above, in some embodiments, sulfurization occurs underconditions which cleave the chiral reagent from the growingoligonucleotide. In some embodiments, sulfurization occurs underconditions which do not cleave the chiral reagent from the growingoligonucleotide.

In some embodiments, a sulfurization reagent is dissolved in a suitablesolvent and delivered to the column. In certain embodiments, the solventis a polar aprotic solvent such as a nitrile solvent. In someembodiments, the solvent is acetonitrile. In some embodiments, asolution of sulfurization reagent is prepared by mixing a sulfurizationreagent (e.g., a thiosulfonate derivative as described herein) withBSTFA (N,O-bis-trimethylsilyl-trifluoroacetamide) in a nitrile solvent(e.g., acetonitrile). In some embodiments, BSTFA is not included. Forexample, the present inventors have found that relatively more reactivesulfurization reagents of general formula R^(s2)—S—S(O)₂—R^(s3) canoften successfully participate in sulfurization reactions in the absenceof BSTFA. To give but one example, the inventors have demonstrated thatwhere R^(s2) is p-nitrophenyl and R^(s3) is methyl then no BSTFA isrequired. In light of this disclosure, those skilled in the art willreadily be able to determine other situations and/or sulfurizationreagents that do not require BSTFA.

In some embodiments, the sulfurization step is performed at roomtemperature. In some embodiments, the sulfurization step is performed atlower temperatures such as about 0° C., about 5° C., about 10° C., orabout 15° C. In some embodiments, the sulfurization step is performed atelevated temperatures of greater than about 20° C.

In some embodiments, a sulfurization reaction is run for about 1 minuteto about 120 minutes. In some embodiments, a sulfurization reaction isrun for about 1 minute to about 90 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 60 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 30 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 25 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 20 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 10 minutes. In some embodiments, asulfurization reaction is run for about 5 minute to about 60 minutes.

In some embodiments, a sulfurization reaction is run for about 5minutes. In some embodiments, a sulfurization reaction is run for about10 minutes. In some embodiments, a sulfurization reaction is run forabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 20 minutes. In some embodiments, a sulfurization reaction isrun for about 25 minutes. In some embodiments, a sulfurization reactionis run for about 30 minutes. In some embodiments, a sulfurizationreaction is run for about 35 minutes. In some embodiments, asulfurization reaction is run for about 40 minutes. In some embodiments,a sulfurization reaction is run for about 45 minutes. In someembodiments, a sulfurization reaction is run for about 50 minutes. Insome embodiments, a sulfurization reaction is run for about 55 minutes.In some embodiments, a sulfurization reaction is run for about 60minutes.

It was unexpectedly found that certain of the sulfurization modificationproducts made in accordance with methods of the present disclosure areunexpectedly stable. In some embodiments, it the unexpectedly stableproducts are phosphorothioate triesters. In some embodiments, theunexpectedly stable products are chirally controlled oligonucleotidescomprising one or more internucleotidic linkages having the structure offormula I-c.

One of skill in the relevant arts will recognize that sulfurizationmethods described herein and sulfurization reagents described herein arealso useful in the context of modifying H-phosphonate oligonucleotidessuch as those described in Wada II (WO2010/064146).

In some embodiments, the sulfurization reaction has a stepwisesulfurization efficiency that is at least about 80%, 85%, 90%, 95%, 96%,97%, or 98%. In some embodiments, the sulfurization reaction provides acrude dinucleotide product composition that is at least 98% pure. Insome embodiments, the sulfurization reaction provides a crudetetranucleotide product composition that is at least 90% pure. In someembodiments, the sulfurization reaction provides a crudedodecanucleotide product composition that is at least 70% pure. In someembodiments, the sulfurization reaction provides a crude icosanucleotideproduct composition that is at least 50% pure.

Once the step of modifying the linkage phosphorus is complete, theoligonucleotide undergoes another deblock step in preparation forre-entering the cycle. In some embodiments, a chiral auxiliary remainsintact after sulfurization and is deblocked during the subsequentdeblock step, which necessarily occurs prior to re-entering the cycle.The process of deblocking, coupling, capping, and modifying, arerepeated until the growing oligonucleotide reaches a desired length, atwhich point the oligonucleotide can either be immediately cleaved fromthe solid support or left attached to the support for purificationpurposes and later cleaved. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in a single step. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in more than one step. In some embodiments, deprotection andcleavage from the support occurs under basic conditions using, e.g., oneor more amine bases. In certain embodiments, the one or more amine basescomprise propyl amine. In certain embodiments, the one or more aminebases comprise pyridine.

In some embodiments, cleavage from the support and/or deprotectionoccurs at elevated temperatures of about 30° C. to about 90° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 40° C. to about 80° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 50° C. to about 70° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 60° C. In some embodiments, cleavage fromthe support and/or deprotection occurs at ambient temperatures.

Example purification procedures are described herein and/or are knowngenerally in the relevant arts.

Noteworthy is that the removal of the chiral auxiliary from the growingoligonucleotide during each cycle is beneficial for at least the reasonsthat (1) the auxiliary will not have to be removed in a separate step atthe end of the oligonucleotide synthesis when potentially sensitivefunctional groups are installed on phosphorus; and (2) unstablephosphorus-auxiliary intermediates prone to undergoing side reactionsand/or interfering with subsequent chemistry are avoided. Thus, removalof the chiral auxiliary during each cycle makes the overall synthesismore efficient.

While the step of deblocking in the context of the cycle is describedabove, additional general methods are included below.

Deblocking Step

In some embodiments, the step of coupling is preceded by a step ofdeblocking. For instance, in some embodiments, the 5′ hydroxyl group ofthe growing oligonucleotide is blocked (i.e., protected) and must bedeblocked in order to subsequently react with a nucleoside couplingpartner.

In some embodiments, acidification is used to remove a blocking group.In some embodiments, the acid is a Brønsted acid or Lewis acid. UsefulBrønsted acids are carboxylic acids, alkylsulfonic acids, arylsulfonicacids, phosphoric acid and its derivatives, phosphonic acid and itsderivatives, alkylphosphonic acids and their derivatives, arylphosphonicacids and their derivatives, phosphinic acid, dialkylphosphinic acids,and diarylphosphinic acids which have a pKa (25° C. in water) value of−0.6 (trifluoroacetic acid) to 4.76 (acetic acid) in an organic solventor water (in the case of 80% acetic acid). The concentration of the acid(1 to 80%) used in the acidification step depends on the acidity of theacid. Consideration to the acid strength must be taken into account asstrong acid conditions will result in depurination/depyrimidination,wherein purinyl or pyrimidinyl bases are cleaved from ribose ring and orother sugar ring. In some embodiments, an acid is selected fromR^(a1)COOH, R^(a1)SO₃H, R^(a3)SO₃H,

wherein each of R^(a1) and R^(a2) is independently hydrogen or anoptionally substituted alkyl or aryl, and R^(a3) is an optionallysubstituted alkyl or aryl.

In some embodiments, acidification is accomplished by a Lewis acid in anorganic solvent. Examples of such useful Lewis acids are Zn(X^(a))₂wherein X^(a) is Cl, Br, I, or CF₃SO₃.

In some embodiments, the step of acidifying comprises adding an amountof a Brønsted or Lewis acid effective to remove a blocking group withoutremoving purine moieties from the condensed intermediate.

Acids that are useful in the acidifying step also include, but are notlimited to 10% phosphoric acid in an organic solvent, 10% hydrochloricacid in an organic solvent, 1% trifluoroacetic acid in an organicsolvent, 3% dichloroacetic acid or trichloroacetic acid in an organicsolvent or 80% acetic acid in water. The concentration of any Brønstedor Lewis acid used in this step is selected such that the concentrationof the acid does not exceed a concentration that causes cleavage of anucleobase from a sugar moiety.

In some embodiments, acidification comprises adding 1% trifluoroaceticacid in an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 8% trifluoroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 3%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding about 0.1% to about 10%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding 3% trichloroacetic acidin an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 10% trichloroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 80% aceticacid in water. In some embodiments, acidification comprises adding about50% to about 90%, or about 50% to about 80%, about 50% to about 70%,about 50% to about 60%, about 70% to about 90% acetic acid in water. Insome embodiments, the acidification comprises the further addition ofcation scavengers to an acidic solvent. In certain embodiments, thecation scavengers can be triethylsilane or triisopropylsilane. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 1% trifluoroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% dichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in dichloromethane.

In certain embodiments, methods of the present disclosure are completedon a synthesizer and the step of deblocking the hydroxyl group of thegrowing oligonucleotide comprises delivering an amount solvent to thesynthesizer column, which column contains a solid support to which theoligonucleotide is attached. In some embodiments, the solvent is ahalogenated solvent (e.g., dichloromethane). In certain embodiments, thesolvent comprises an amount of an acid. In some embodiments, the solventcomprises an amount of an organic acid such as, for instance,trichloroacetic acid. In certain embodiments, the acid is present in anamount of about 1% to about 20% w/v. In certain embodiments, the acid ispresent in an amount of about 1% to about 10% w/v. In certainembodiments, the acid is present in an amount of about 1% to about 5%w/v. In certain embodiments, the acid is present in an amount of about 1to about 3% w/v. In certain embodiments, the acid is present in anamount of about 3% w/v. Methods for deblocking a hydroxyl group aredescribed further herein. In some embodiments, the acid is present in 3%w/v is dichloromethane.

In some embodiments, the chiral auxiliary is removed before thedeblocking step. In some embodiments, the chiral auxiliary is removedduring the deblocking step.

In some embodiments, cycle exit is performed before the deblocking step.In some embodiments, cycle exit is preformed after the deblocking step.

General Conditions for Blocking Group/Protecting Group Removal

Functional groups such as hydroxyl or amino moieties which are locatedon nucleobases or sugar moieties are routinely blocked with blocking(protecting) groups (moieties) during synthesis and subsequentlydeblocked. In general, a blocking group renders a chemical functionalityof a molecule inert to specific reaction conditions and can later beremoved from such functionality in a molecule without substantiallydamaging the remainder of the molecule (see e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, NewYork, 1991). For example, amino groups can be blocked with nitrogenblocking groups such as phthalimido, 9-fluorenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC, 4,4′-dimethoxytrityl (DMTr),4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be protectedas acetyl groups. Hydroxy groups can be protected such astetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),1-(2-chloroethoxy)ethyl, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDP),bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl (TOM),1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl (CN),pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representativehydroxyl blocking groups have been described (see e.g., Beaucage et al.,Tetrahedron, 1992, 46, 2223). In some embodiments, hydroxyl blockinggroups are acid-labile groups, such as the trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups canalso be blocked by including them in a precursor form. Thus an azidogroup can be considered a blocked form of an amine as the azido group iseasily converted to the amine. Further representative protecting groupsutilized in nucleic acid synthesis are known (see e.g. Agrawal et al.,Protocols for Oligonucleotide Conjugates, Eds., Humana Press, NewJersey, 1994, Vol. 26, pp. 1-72).

Various methods are known and used for removal of blocking groups fromnucleic acids. In some embodiments, all blocking groups are removed. Insome embodiments, a portion of blocking groups are removed. In someembodiments, reaction conditions can be adjusted to selectively removecertain blocking groups.

In some embodiments, nucleobase blocking groups, if present, arecleavable with an acidic reagent after the assembly of a providedoligonucleotide. In some embodiment, nucleobase blocking groups, ifpresent, are cleavable under neither acidic nor basic conditions, e.g.cleavable with fluoride salts or hydrofluoric acid complexes. In someembodiments, nucleobase blocking groups, if present, are cleavable inthe presence of base or a basic solvent after the assembly of a providedoligonucleotide. In certain embodiments, one or more of the nucleobaseblocking groups are characterized in that they are cleavable in thepresence of base or a basic solvent after the assembly of a providedoligonucleotide but are stable to the particular conditions of one ormore earlier deprotection steps occurring during the assembly of theprovided oligonucleotide.

In some embodiments, blocking groups for nucleobases are not required.In some embodiments, blocking groups for nucleobases are required. Insome embodiments, certain nucleobases require one or more blockinggroups while other nucleobases do not require one or more blockinggroups.

In some embodiments, the oligonucleotide is cleaved from the solidsupport after synthesis. In some embodiments, cleavage from the solidsupport comprises the use of propylamine. In some embodiments, cleavagefrom the solid support comprises the use of propylamine in pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in pyridine. In some embodiments, cleavage from thesolid support comprises the use of propylamine in anhydrous pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in anhydrous pyridine. In some embodiments, cleavagefrom the solid support comprises use of a polar aprotic solvent such asacetonitrile, NMP, DMSO, sulfone, and/or lutidine. In some embodiments,cleavage from the solid support comprises use of solvent, e.g., a polaraprotic solvent, and one or more primary amines (e.g., a C₁₋₁₀ amine),and/or one or more of methoxylamine, hydrazine, and pure anhydrousammonia.

In some embodiments, deprotection of oligonucleotide comprises the useof propylamine. In some embodiments, deprotection of oligonucleotidecomprises the use of propylamine in pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inpyridine. In some embodiments deprotection of oligonucleotide comprisesthe use of propylamine in anhydrous pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inanhydrous pyridine.

In some embodiments, the oligonucleotide is deprotected during cleavage.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about room temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at elevated temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at above about 30° C., 40°C., 50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about 30° C., 40° C.,50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 40-80° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 50-70° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 60° C.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for more than 0.1 hr, 1hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 0.1-5 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 3-10 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5-15 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10-20 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15-25 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 20-40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 2 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 18 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 24 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature formore than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30hrs, or 40 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed at roomtemperature for about 5-48 hrs. In some embodiments, cleavage ofoligonucleotide from solid support, or deprotection of oligonucleotide,is performed at room temperature for about 10-24 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature forabout 18 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed atelevated temperature for more than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs,15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. In some embodiments, cleavageof oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at elevated temperature for about 0.5-5hrs. In some embodiments, cleavage of oligonucleotide from solidsupport, or deprotection of oligonucleotide, is performed at about 60°C. for about 0.5-5 hrs. In some embodiments, cleavage of oligonucleotidefrom solid support, or deprotection of oligonucleotide, is performed atabout 60° C. for about 2 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide comprises the use of propylamine and isperformed at room temperature or elevated temperature for more than 0.1hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40hrs. Example conditions are 20% propylamine in pyridine at roomtemperature for about 18 hrs, and 20% propylamine in pyridine at 60° C.for about 18 hrs,

In some embodiments, prior to cleavage from solid support, a step isperformed to remove a chiral auxiliary group, if one is still attachedto an internucleotidic phosphorus atom. In some embodiments, forexample, one or more DPSE type chiral auxiliary groups remain attachedto internucleotidic phosphorus atoms during the oligonucleotidesynthesis cycle. Suitable conditions for removing remaining chiralauxiliary groups are widely known in the art, e.g., those described inWada I, Wada II, Wada III, Chiral Control, etc. In some embodiments, acondition for removing DPSE type chiral auxiliary is TBAF or HF-Et₃N,e.g., 0.1M TBAF in MeCN, 0.5M HF-Et₃N in THF or MeCN, etc. In someembodiments, the present disclosure recognizes that a linker may becleaved during the process of removing a chiral auxiliary group. In someembodiments, the present disclosure provides linkers, such as the SPlinker, that provides better stability during chiral auxiliary groupremoval. Among other things, certain linkers provided by the presentdisclosure provided improved yield and/or purity.

In some embodiments, an activator is a “Wada” activator, i.e., theactivator is from any one of Wada I, II, or III documents cited above.

Example activating groups are depicted below:

In some embodiments, an activating reagent is selected from

In some embodiments, an example cycle is depicted in Scheme I-b.

In some embodiments, an example cycle is illustrated in Scheme I-c.

In Scheme I-c, oligonucleotide (or nucleotide, or oligonucleotide withmodified internucleotidic linkage) on solid support (C-1) is coupledwith phosphoramidite C-2. After coupling and capping, an oxidation stepis performed. After deblocking, a phosphate diester linkage is formed.The cycle product C-3 can either re-enter cycle C to install morephosphate diester linkage, or enter other cycles to install other typesof internucleotidic linkages, or go to cycle exit.

In some embodiments, non-chirally pure phosphoramidite can be usedinstead of C-2 in Scheme I-c. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, the use of a phosphorothioate diester precursorincreases the stability of oligonucleotide during synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe efficiency of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe yield of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe product purity of chirally controlled oligonucleotide synthesis.

In some embodiments, the phosphorothioate diester precursor in theabove-mentioned methods is

In some embodiments,

is converted to a phosphorothioate diester linkage duringdeprotection/release. In some embodiments, an example cycle is depictedin Scheme I-d. More examples are depicted below.

As illustrated in Scheme I-d, both phosphorothioate and phosphatediester linkages can be incorporated into the same chirally controlledoligonucleotide. As understood by a person of ordinary skill in the art,the provided methods do not require that the phosphorothioate diesterand the phosphate diester to be consecutive—other internucleotidiclinkages can form between them using a cycle as described above. InScheme I-d, phosphorothioate diester precursors,

are installed in place of the phosphorothioate diester linkages. In someembodiments, such replacement provided increased synthesis efficiencyduring certain steps, for instance, the oxidation step. In someembodiments, the use of phosphorothioate diester precursors generallyimprove the stability of chirally controlled oligonucleotides duringsynthesis and/or storage. After cycle exit, during deprotection/release,the phosphorothioate diester precursor is converted to phosphorothioatediester linkage. In some embodiments, it is beneficial to usephosphorothioate diester precursor even when no phosphate diesterlinkage is present in the chirally controlled oligonucleotide, or nooxidation step is required during synthesis.

As in Scheme I-c, in some embodiments, non-chirally pure phosphoramiditecan be used for cycles comprising oxidation steps. In some embodiments,β-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, methods of the present disclosure provide chirallycontrolled oligonucleotide compositions that are enriched in aparticular oligonucleotide type.

In some embodiments, at least about 10% of a provided crude compositionis of a particular oligonucleotide type. In some embodiments, at leastabout 20% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 30% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 50% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 60% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 70% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 80% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 90% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided crude composition isof a particular oligonucleotide type.

In some embodiments, at least about 1% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 2%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 3% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 4%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 5% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 10%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 20% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 30%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 50%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 60% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 70%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 80% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 90%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided composition is of aparticular oligonucleotide type.

In some embodiments, an example cycle is depicted in Scheme I-e, below.

In some embodiments, X is H or a 2′-modification. In some embodiments, Xis H or —OR¹, wherein R¹ is not hydrogen. In some embodiments, X is H or—OR¹, wherein R¹ is optionally substituted C₁₋₆ alkyl. In someembodiments, X is H. In some embodiments, X is —OMe. In someembodiments, X is —OCH₂CH₂OCH₃. In some embodiments, X is —F.

In some embodiments, an example cycle is depicted in Scheme I-f.

In some embodiments, X is H or a 2′-modification. In some embodiments, Xis H or —OR¹, wherein R¹ is not hydrogen. In some embodiments, X is H or—OR¹, wherein R¹ is optionally substituted C₁₋₆ alkyl. In someembodiments, X is H. In some embodiments, X is —OMe. In someembodiments, X is —OCH₂CH₂OCH₃. In some embodiments, X is —F.

It is understood by a person having ordinary skill in the art thatdifferent types of cycles may be combined to provide complete control ofthe chemical modifications and stereochemistry of oligonucleotides. Insome embodiments, for example, an oligonucleotide synthesis process maycontain one or more Cycles A-F. In some embodiments, a provided methodcomprises at least one cycle using a DPSE-type chiral auxiliary.

In some embodiments, the present disclosure provides methods forpreparing provided oligonucleotide and oligonucleotide compositions. Insome embodiments, a provide methods comprising providing a providedchiral reagent having the structure of

wherein W¹ is —NG⁵, W² is O, each of G¹ and G³ is independently hydrogenor an optionally substituted group selected from C₁₋₁₀ aliphatic,heterocyclyl, heteroaryl and aryl, G² is —C(R)₂Si(R)₃, and G⁴ and G⁵ aretaken together to form an optionally substituted saturated, partiallyunsaturated or unsaturated heteroatom-containing ring of up to about 20ring atoms which is monocyclic or polycyclic, fused or unfused, whereineach R is independently hydrogen, or an optionally substituted groupselected from C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, andheterocyclyl. In some embodiments, a provided chiral reagent has thestructure of

In some embodiments, a provided methods comprises providing aphosphoramidite comprising a moiety from a chiral reagent having thestructure of

wherein —W¹H and —W²H, or the hydroxyl and amino groups, form bonds withthe phosphorus atom of the phosphoramidite. In some embodiments, —W¹Hand —W²H, or the hydroxyl and amino groups, form bonds with thephosphorus atom of the phosphoramidite, e.g., in

In some embodiments, a phosphoramidite has the structure of

In some embodiments, R is a protection group. In some embodiments, R isDMTr. In some embodiments, G² is —C(R)₂Si(R)₃, wherein —C(R)₂— isoptionally substituted —CH₂—, and each R of —Si(R)₃ is independently anoptionally substituted group selected from C₁₋₁₀ aliphatic,heterocyclyl, heteroaryl and aryl. In some embodiments, at least one Rof —Si(R)₃ is independently optionally substituted C₁₋₁₀ alkyl. In someembodiments, at least one R of —Si(R)₃ is independently optionallysubstituted phenyl. In some embodiments, one R of —Si(R)₃ isindependently optionally substituted phenyl, and each of the other two Ris independently optionally substituted C₁₋₁₀ alkyl. In someembodiments, one R of —Si(R)₃ is independently optionally substitutedC₁₋₁₀ alkyl, and each of the other two R is independently optionallysubstituted phenyl. In some embodiments, G² is optionally substituted—CH₂Si(Ph)(Me)₂. In some embodiments, G² is optionally substituted—CH₂Si(Me)(Ph)₂. In some embodiments, G² is —CH₂Si(Me)(Ph)₂. In someembodiments, G⁴ and G⁵ are taken together to form an optionallysubstituted saturated 5-6 membered ring containing one nitrogen atom (towhich G⁵ is attached). In some embodiments, G⁴ and G⁵ are taken togetherto form an optionally substituted saturated 5-membered ring containingone nitrogen atom. In some embodiments, G¹ is hydrogen. In someembodiments, G³ is hydrogen. In some embodiments, both G¹ and G³ arehydrogen. In some embodiments, both G¹ and G³ are hydrogen, G² is—C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, and eachR of —Si(R)₃ is independently an optionally substituted group selectedfrom C₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl, and G⁴ and G⁵are taken together to form an optionally substituted saturated5-membered ring containing one nitrogen atom. In some embodiments, aprovided method further comprises providing a fluoro-containing reagent.In some embodiments, a provided fluoro-containing reagent removes achiral reagent, or a product formed from a chiral reagent, fromoligonucleotides after synthesis. Various known fluoro-containingreagents, including those F sources for removing —SiR₃ groups, can beutilized in accordance with the present disclosure, for example, TBAF,HF₃-Et₃N etc. In some embodiments, a fluoro-containing reagent providesbetter results, for example, shorter treatment time, lower temperature,less desulfurization, etc, compared to traditional methods, such asconcentrated ammonia. In some embodiments, for certain fluoro-containingreagent, the present disclosure provides linkers for improved results,for example, less cleavage of oligonucleotides from support duringremoval of chiral reagent (or product formed therefrom duringoligonucleotide synthesis). In some embodiments, a provided linker is anSP linker. In some embodiments, the present disclosure demonstrated thata HF-base complex can be utilized, such as HF—NR₃, to control cleavageduring removal of chiral reagent (or product formed therefrom duringoligonucleotide synthesis). In some embodiments, HF—NR₃ is HF—NEt₃. Insome embodiments, HF—NR₃ enables use of traditional linkers, e.g.,succinyl linker.

Biological Applications and Example of Use

Among other things, the present disclosure recognizes that propertiesand activities of an oligonucleotide can be adjusted by optimizing itspattern of backbone chiral centers through the use of provided chirallycontrolled oligonucleotide compositions. In some embodiments, thepresent disclosure provides chirally controlled oligonucleotidecompositions, wherein the oligonucleotides have a common pattern ofbackbone chiral centers which enhances their stability and/or biologicalactivity. In some embodiments, a pattern of backbone chiral centersprovides unexpectedly increased stability. In some embodiments, apattern of backbone chiral centers, surprisingly, provides greatlyincreased activity. In some embodiments, a pattern of backbone chiralcenters provides both increased stability and activity. In someembodiments, when an oligonucleotide is utilized to cleave a nucleicacid polymer, a pattern of backbone chiral centers of theoligonucleotide, surprisingly by itself, changes the cleavage pattern ofa target nucleic acid polymer. In some embodiments, a pattern ofbackbone chiral centers effectively prevents cleavage at secondarysites. In some embodiments, a pattern of backbone chiral centers createsnew cleavage sites. In some embodiments, a pattern of backbone chiralcenters minimizes the number of cleavage sites. In some embodiments, apattern of backbone chiral centers minimizes the number of cleavagesites so that a target nucleic acid polymer is cleaved at only one sitewithin the sequence of the target nucleic acid polymer that iscomplementary to the oligonucleotide. In some embodiments, a pattern ofbackbone chiral centers enhances cleavage efficiency at a cleavage site.In some embodiments, a pattern of backbone chiral centers of theoligonucleotide improves cleavage of a target nucleic acid polymer. Insome embodiments, a pattern of backbone chiral centers increasesselectivity. In some embodiments, a pattern of backbone chiral centersminimizes off-target effect. In some embodiments, a pattern of backbonechiral centers increase selectivity, e.g., cleavage selectivity amongtarget sequences differing by point mutations or single nucleotidepolymorphisms (SNPs). In some embodiments, a pattern of backbone chiralcenters increase selectivity, e.g., cleavage selectivity among targetsequences differing by only one point mutation or single nucleotidepolymorphism (SNP).

Among other things, it is surprisingly found that certain providedoligonucleotide compositions achieve unprecedented control of cleavageof target sequences, e.g., cleavage of target RNA by RNase H. In someembodiments, the present disclosure demonstrates that precise control ofchemical and stereochemical attributes of oligonucleotides achievesimproved activity of oligonucleotide preparations as compared withotherwise comparable preparations for which stereochemical attributesare not controlled. Among other things, the present disclosurespecifically demonstrates improved rate, degree, and or specificity ofcleavage of nucleic acid targets to which provided oligonucleotideshybridize.

In some embodiments, the present disclosure provides various uses ofoligonucleotide compositions. Among other things, the present disclosuredemonstrates that by controlling structural elements ofoligonucleotides, such as base sequence, chemical modifications,stereochemistry, etc., properties of oligonucleotides can be greatlyimproved. For example, in some embodiments, the present disclosureprovides methods for highly selective suppression of transcripts of atarget nucleic acid sequence. In some embodiments, the presentdisclosure provides methods for treating a subject by suppressingtranscripts from a diseasing-causing copy (e.g., a disease-causingallele). In some embodiments, the present disclosure provides methodsfor designing and preparing oligonucleotide compositions withsurprisingly enhanced activity and/or selectivity when suppressing atranscript of a target sequence. In some embodiments, the presentdisclosure provides methods for designing and/or preparingoligonucleotide compositions which provide allele-specific suppressionof a transcript from a target nucleic acid sequence.

In some embodiments, the present disclosure provides a method forcontrolled cleavage of a nucleic acid polymer, the method comprisingsteps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type.

In some embodiments, the present disclosure provides a method foraltering a cleavage pattern observed when a nucleic acid polymer whosenucleotide sequence includes a target sequence is contacted with areference oligonucleotide composition that comprises oligonucleotideshaving a particular base sequence and length, which particular basesequence is or comprises a sequence that is complementary to the targetsequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

-   -   1) the particular base sequence and length;    -   2) a particular pattern of backbone linkages; and    -   3) a particular pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a method forcontrolled cleavage of a nucleic acid polymer, comprising providing achirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers; and        wherein the nucleic acid polymer is cleaved in a cleavage        pattern that is different than the cleavage pattern when        chirally uncontrolled oligonucleotide composition is provided.

As used herein, a cleavage pattern of a nucleic acid polymer is definedby the number of cleavage sites, the locations of the cleavage sites,and the percentage of cleavage at each sites. In some embodiments, acleavage pattern has multiple cleavage sites, and the percentage ofcleavage at each site is different. In some embodiments, a cleavagepattern has multiple cleavage sites, and the percentage of cleavage ateach site is the same. In some embodiments, a cleavage pattern has onlyone cleavage site. In some embodiments, cleavage patterns differ fromeach other in that they have different numbers of cleavage sites. Insome embodiments, cleavage patterns differ from each other in that atleast one cleavage location is different. In some embodiments, cleavagepatterns differ from each other in that the percentage of cleavage atleast one common cleavage site is different. In some embodiments,cleavage patterns differ from each other in that they have differentnumbers of cleavage sites, and/or at least one cleavage location isdifferent, and/or the percentage of cleavage at least one commoncleavage site is different.

In some embodiments, the present disclosure provides a method forcontrolled cleavage of a nucleic acid polymer, the method comprisingsteps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type, the        contacting being performed under conditions so that cleavage of        the nucleic acid polymer occurs.

In some embodiments, the present disclosure provides a method forchanging a first cleavage pattern of a nucleic acid polymer resultedfrom using a first oligonucleotide composition, comprising providing asecond chirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers; and        wherein the nucleic acid polymer is cleaved in a cleavage        pattern that is different than the first cleavage pattern.

In some embodiments, the present disclosure provides a method foraltering a cleavage pattern observed when a nucleic acid polymer whosenucleotide sequence includes a target sequence is contacted with areference oligonucleotide composition that comprises oligonucleotideshaving a particular base sequence and length, which particular basesequence is or comprises a sequence that is complementary to the targetsequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

1) the particular base sequence and length;

2) a particular pattern of backbone linkages; and

3) a particular pattern of backbone chiral centers,

the contacting being performed under conditions so that cleavage of thenucleic acid polymer occurs.

In some embodiments, a provided chirally controlled oligonucleotidecomposition reduces the number of cleavage sites within the targetsequence. In some embodiments, a provided chirally controlledoligonucleotide composition provides single-site cleavage within thetarget sequence. In some embodiments, a chirally controlledoligonucleotide composition provides enhanced cleavage rate at acleavage site within the target sequence. In some embodiments, achirally controlled oligonucleotide composition provides enhancedefficiency at a cleavage site within the target sequence. In someembodiments, a chirally controlled oligonucleotide composition providesincreased turn-over in cleaving a target nucleic acid polymer. In someembodiments, a chirally controlled oligonucleotide composition increasepercentage of cleavage at a site within or in the vicinity of acharacteristic sequence element. In some embodiments, a chirallycontrolled oligonucleotide composition increase percentage of cleavageat a site in the vicinity of a mutation. In some embodiments, a chirallycontrolled oligonucleotide composition increase percentage of cleavageat a site in the vicinity of a SNP. Example embodiments of a site withinor in the vicinity of a characteristic sequence element, in the vicinityof a mutation, in the vicinity of a SNP, are described in the presentdisclosure. For example, in some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, 2, 3, 4, or 5 internucleotidiclinkages away from a mutation; in some other embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, 3, 4, or 5internucleotidic linkages away from a SNP.

In some embodiments, cleavage occurs with a cleavage pattern differsfrom a reference cleavage pattern. In some embodiments, a referencecleavage pattern is one observed when a nucleic acid polymer iscontacted under comparable conditions with a reference oligonucleotidecomposition. In some embodiments, a reference oligonucleotidecomposition is a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of oligonucleotides that share the commonbase sequence and length of a chirally controlled oligonucleotidecomposition. In some embodiments, a reference oligonucleotidecomposition is a substantially racemic preparation of oligonucleotidesthat share the common sequence and length.

In some embodiments, a nucleic acid polymer is RNA. In some embodiments,a nucleic acid polymer is an oligonucleotide. In some embodiments, anucleic acid polymer is an RNA oligonucleotide. In some embodiments, anucleic acid polymer is a transcript. In some embodiments,oligonucleotides of a provided chirally controlled oligonucleotidecomposition form duplexes with a nucleic acid polymer to be cleaved.

In some embodiments, a nucleic acid polymer is cleaved by an enzyme. Insome embodiments, an enzyme cleaves a duplex formed by a nucleic acidpolymer. In some embodiments, an enzyme is RNase H. In some embodiments,an enzyme is Dicer. In some embodiments, an enzyme is an Argonauteprotein. In some embodiments, an enzyme is Ago2. In some embodiments, anenzyme is within a protein complex. An example protein complex isRNA-induced silencing complex (RISC).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprising oligonucleotides with a common pattern ofbackbone chiral centers provides unexpectedly high selectivity so thatnucleic acid polymers that have only small sequence variations within atarget region can be selectively targeted. In some embodiments, anucleic acid polymer is a transcript from an allele. In someembodiments, transcripts from different alleles can be selectivelytargeted by provided chirally controlled oligonucleotide compositions.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods thereof enables precise control of cleavagesites within a target sequence. In some embodiments, a cleavage site isaround a sequence of RpSpSp backbone chiral centers. In someembodiments, a cleavage site is upstream of and near a sequence ofRpSpSp backbone chiral centers. In some embodiments, a cleavage site iswithin 5 base pairs upstream of a sequence of RpSpSp backbone chiralcenters. In some embodiments, a cleavage site is within 4 base pairsupstream of a sequence of RpSpSp backbone chiral centers. In someembodiments, a cleavage site is within 3 base pairs upstream of asequence of RpSpSp backbone chiral centers. In some embodiments, acleavage site is within 2 base pairs upstream of a sequence of RpSpSpbackbone chiral centers. In some embodiments, a cleavage site is within1 base pair upstream of a sequence of RpSpSp backbone chiral centers. Insome embodiments, a cleavage site is downstream of and near a sequenceof RpSpSp backbone chiral centers. In some embodiments, a cleavage siteis within 5 base pairs downstream of a sequence of RpSpSp backbonechiral centers. In some embodiments, a cleavage site is within 4 basepairs downstream of a sequence of RpSpSp backbone chiral centers. Insome embodiments, a cleavage site is within 3 base pairs downstream of asequence of RpSpSp backbone chiral centers. In some embodiments, acleavage site is within 2 base pairs downstream of a sequence of RpSpSpbackbone chiral centers. In some embodiments, a cleavage site is within1 base pair downstream of a sequence of RpSpSp backbone chiral centers.Among other things, the present disclosure therefore provides control ofcleavage sites with in a target sequence. In some embodiments, anexample cleavage is depicted in FIG. 21. In some embodiments, cleavagedepicted in FIG. 21 is designated as cleavage at a site two base pairsdownstream a sequence of RpSpSp backbone chiral centers. As extensivelydescribed in the present disclosure, a sequence of RpSpSp backbonechiral centers can be found in a single or repeating units of(Np)_(m)(Rp)_(n)(Sp)_(t), (Np)_(t)(Rp)_(n)(Sp)_(m),(Sp)_(m)(Rp)_(n)(Sp)_(t), (Sp)_(t)(Rp)_(n)(Sp)_(m), (Rp)_(n)(Sp)_(m),(Rp)_(m)(Sp)_(n), (Sp)_(m)Rp and/or Rp(Sp)_(m), each of which isindependently as defined above and described herein. In someembodiments, a provided chirally controlled oligonucleotide compositioncreates a new cleavage site 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule (e.g., see FIG. 21), wherein saidnew cleavage site does not exist if a reference (e.g., chirallyuncontrolled) oligonucleotide composition is used (cannot be detected).In some embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at a cleavage site 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21), wherein cleavage at such a site occurs at a higher percentage thanwhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, cleavage at such a site by aprovided chirally controlled oligonucleotide composition is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500or 1000 fold of that by a reference oligonucleotide composition (forexample, when measured by percentage of cleavage at a site). In someembodiments, a provided chirally controlled oligonucleotide compositionprovides accelerated cleavage at a cleavage site 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21), compared to when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, cleavage by aprovided chirally controlled oligonucleotide composition is at least 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500or 1000 fold faster than that by a reference oligonucleotidecomposition. In some embodiments, a cleavage site of a provided chirallycontrolled oligonucleotide composition 2 base pairs downstream of RpSpSpbackbone chiral centers in a target molecule (e.g., see FIG. 21) is acleavage site when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, a cleavagesite of a provided chirally controlled oligonucleotide composition 2base pairs downstream of RpSpSp backbone chiral centers in a targetmolecule (e.g., see FIG. 21) is within one base pair of a cleavage sitewhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, a cleavage site of a providedchirally controlled oligonucleotide composition 2 base pairs downstreamof RpSpSp backbone chiral centers in a target molecule (e.g., see FIG.21) is within 2 base pairs of a cleavage site when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used. In someembodiments, it is within 3 base pairs. In some embodiments, it iswithin 4 base pairs. In some embodiments, it is within 5 base pairs. Insome embodiments, a cleavage site of a provided chirally controlledoligonucleotide composition 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule is one of the major cleavage siteswhen a reference (e.g., chirally uncontrolled) oligonucleotidecomposition is used. In some embodiments, such a site is the cleavagesite with the highest cleavage percentage when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used. In someembodiments, a cleavage site of a provided chirally controlledoligonucleotide composition 2 base pairs downstream of RpSpSp backbonechiral centers in a target molecule is one of the cleavage sites withhigher cleavage rate when a reference (e.g., chirally uncontrolled)oligonucleotide composition is used. In some embodiments, such a site isthe cleavage site with the highest cleavage rate when a reference (e.g.,chirally uncontrolled) oligonucleotide composition is used.

In some embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at one or more sites, e.g., relative to areference (e.g., chirally uncontrolled/stereorandom) oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition selectively enhances cleavage at a singlesite relative to a reference (e.g., chirally uncontrolled/stereorandom)composition. In some embodiments, a chirally controlled oligonucleotidecomposition enhances cleavage at a site by providing a higher cleavagerate. In some embodiments, a chirally controlled oligonucleotidecomposition enhances cleavage at a site by providing a higher percentageof cleavage at said site. Percentage of cleavage at a site can bedetermined by various methods widely known and practiced in the art. Insome embodiments, percentage of cleavage at a site is determined byanalysis of cleavage products, for example, as by HPLC-MS as illustratedin FIG. 18, FIG. 19 and FIG. 30; see also example cleavage maps such asFIG. 9, FIG. 10, FIG. 11, FIG. 14, FIG. 22, FIG. 25 and FIG. 26. In someembodiments, enhancement is relative to a reference oligonucleotidecomposition. In some embodiments, enhancement is relative to anothercleavage site. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site that is apreferred cleavage site of a reference oligonucleotide composition. Insome embodiments, a preferred cleavage site, or a group of preferredcleavage sites, is a site or sites that have relatively higherpercentage of cleavage compared to one or more other cleavage sites. Insome embodiments, preferred cleavage sites can indicate preference of anenzyme. For example, for RNase H, when a DNA oligonucleotide is used,resulting cleavage sites may indicate preference of RNase H. In someembodiments, a provided chirally controlled oligonucleotide compositionenhances cleavage at a site that is a preferred cleavage site of anenzyme. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site that is not apreferred cleavage site of a reference oligonucleotide composition. Insome embodiments, a provided chirally controlled oligonucleotidecomposition enhances cleavage at a site that is not a cleavage site of areference oligonucleotide composition, effectively creating a newcleavage site which does not exist when a reference oligonucleotidecomposition is used. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site within 5 basepairs from a targeted mutation or SNP, thereby increasing selectivecleavage of the undesired target oligonucleotide. In some embodiments, aprovided chirally controlled oligonucleotide composition enhancescleavage at a site within 4 base pairs from a targeted mutation or SNP,thereby increasing selective cleavage of the undesired targetoligonucleotide. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site within 3 basepairs from a targeted mutation or SNP, thereby increasing selectivecleavage of the undesired target oligonucleotide. In some embodiments, aprovided chirally controlled oligonucleotide composition enhancescleavage at a site within 2 base pairs from a targeted mutation or SNP,thereby increasing selective cleavage of the undesired targetoligonucleotide. In some embodiments, a provided chirally controlledoligonucleotide composition enhances cleavage at a site immediatelyupstream or downstream targeted mutation or SNP, thereby increasingselective cleavage of the undesired target oligonucleotide (e.g., FIG.22, Panel D, muRNA).

In some embodiments, a provided chirally controlled oligonucleotidecomposition suppresses cleavage at one or more sites, e.g., relative toa reference (e.g., chirally uncontrolled/stereorandom) oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition selectively suppresses cleavage at a singlesite relative to a reference (e.g., chirally uncontrolled/stereorandom)composition. In some embodiments, a chirally controlled oligonucleotidecomposition suppresses cleavage at a site by providing a lower cleavagerate. In some embodiments, a chirally controlled oligonucleotidecomposition suppresses cleavage at a site by providing a lowerpercentage of cleavage at said site. In some embodiments, suppression isrelative to a reference oligonucleotide composition. In someembodiments, suppression is relative to another cleavage site. In someembodiments, a provided chirally controlled oligonucleotide compositionsuppresses cleavage at a site that is a preferred cleavage site of areference oligonucleotide composition. In some embodiments, a preferredcleavage site, or a group of preferred cleavage sites, is a site orsites that have relatively higher percentage of cleavage compared to oneor more other cleavage sites. In some embodiments, preferred cleavagesites can indicate preference of an enzyme. For example, for RNase H,when a DNA oligonucleotide is used, resulting cleavage sites mayindicate preference of RNase H. In some embodiments, a provided chirallycontrolled oligonucleotide composition suppresses cleavage at a sitethat is a preferred cleavage site of an enzyme. In some embodiments, aprovided chirally controlled oligonucleotide composition suppressescleavage at a site that is not a preferred cleavage site of a referenceoligonucleotide composition. In some embodiments, a provided chirallycontrolled oligonucleotide composition suppresses all cleavage sites ofa reference oligonucleotide composition. In some embodiments, a providedchirally controlled oligonucleotide composition generally enhancescleavage of target oligonucleotides. In some embodiments, a providedchirally controlled oligonucleotide composition generally suppressescleavage of non-target oligonucleotides. In some embodiments, a providedchirally controlled oligonucleotide composition enhances cleavage oftarget oligonucleotides and suppresses cleavage of non-targetoligonucleotides. Using FIG. 22, Panel D, as an example, a targetoligonucleotide for cleavage is muRNA, while a non-targetoligonucleotide is wtRNA. In a subject comprising a diseased tissuecomprising a mutation or SNP, a target oligonucleotide for cleavage canbe transcripts with a mutation or SNP, while a non-targetoligonucleotide can be normal transcripts without a mutation or SNP,such as those expressed in healthy tissues.

In some embodiments, a reference oligonucleotide composition is astereorandom oligonucleotide composition. In some embodiments, areference oligonucleotide composition is a stereorandom composition ofoligonucleotides of which all internucleotidic linkages arephosphorothioate. In some embodiments, a reference oligonucleotidecomposition is a DNA oligonucleotide composition with all phosphatelinkages.

In some embodiments, besides patterns of backbone chiral centersdescribed herein, provided oligonucleotides optionally comprisesmodified bases, modified sugars, modified backbone linkages and anycombinations thereof. In some embodiments, a provided oligonucleotide isa unimer, altmer, blockmer, gapmer, hemimer and skipmer. In someembodiments, a provided oligonucleotide comprises one or more unimer,altmer, blockmer, gapmer, hemimer or skipmer moieties, or anycombinations thereof. In some embodiments, besides patterns of backbonechiral centers herein, a provided oligonucleotide is a hemimer. In someembodiments, besides patterns of backbone chiral centers herein, aprovided oligonucleotide is a 5′-hemimer with modified sugar moieties.In some embodiments, a provided oligonucleotide is 5′-hemimer with2′-modified sugar moieties. Suitable modifications are widely known inthe art, e.g., those described in the present application. In someembodiments, a modification is 2′-F. In some embodiments, a modificationis 2′-MOE. In some embodiments, a modification is s-cEt.

In some embodiments, the present disclosure provides a method forsuppression of a transcript from a target nucleic acid sequence forwhich one or more similar nucleic acid sequences exist within apopulation, each of the target and similar sequences contains a specificnucleotide characteristic sequence element that defines the targetsequence relative to the similar sequences, the method comprising stepsof:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

In some embodiments, the present disclosure provides a method forsuppression of a transcript from a target nucleic acid sequence forwhich one or more similar nucleic acid sequences exist within apopulation, each of the target and similar sequences contains a specificnucleotide characteristic sequence element that defines the targetsequence relative to the similar sequences, the method comprising stepsof:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

In some embodiments, a common base sequence is or comprises a sequencethat is 100% complementary to the characteristic sequence elements. Insome embodiments, suppression can be assessed through various suitableassays as known by a person having ordinary skill in the art. In someembodiments, an assay is a RNase H assay as described in the presentdisclosure, which can assess suppression by evaluating cleavage of asequence found in a transcript of a target nucleic acid sequencecomprising the characteristic sequence element and cleavage of asequence found in a transcript of a similar sequence. In someembodiments, transcripts of the target nucleic acid sequence aresuppressed at a greater level than a level of suppression observed forany one of the similar nucleic acid sequence. In some embodiments,example target and similar sequences are described in the presentdisclosure.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence,the contacting being performed under conditions determined to permit thecomposition to suppress transcripts of the particular allele.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target nucleic acidsequence for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget nucleic acid sequence, the method comprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence,

the contacting being performed under conditions determined to permit thecomposition to suppress transcripts of the particular allele.

In some embodiments, a transcript is suppressed by cleavage of saidtranscript. In some embodiments, a specific nucleotide characteristicsequence element is in an intron. In some embodiments, a specificnucleotide characteristic sequence element is in an exon. In someembodiments, a specific nucleotide characteristic sequence element ispartially in an exon and partially in an intron. In some embodiments, aspecific nucleotide characteristic sequence element comprises a mutationthat differentiates an allele from other alleles. In some embodiments, amutation is a deletion. In some embodiments, a mutation is an insertion.In some embodiments, a mutation is a point mutation. In someembodiments, a specific nucleotide characteristic sequence elementcomprises at least one single nucleotide polymorphism (SNP) thatdifferentiates an allele from other alleles.

In some embodiments, a target nucleic acid sequence is a target gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a gene whose sequence comprises at leastone single nucleotide polymorphism (SNP), comprising providing achirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is completely        complementary to a sequence found in a transcript from the first        allele but not to the corresponding sequence found in a        transcript from the second allele, wherein the sequence found in        the transcripts comprises a SNP site;    -   2) a common pattern of backbone linkages;    -   3) a common pattern of backbone chiral centers, which        composition is a substantially pure preparation of a single        oligonucleotide in that at least about 10% of the        oligonucleotides in the composition have the common base        sequence and length, the common pattern of backbone linkages,        and the common pattern of backbone chiral centers;        wherein the transcript from the first allele is suppressed at        least five folds more than that from the second allele.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene,the contacting being performed under conditions determined to permit thecomposition to suppress transcripts of the particular allele.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene,the contacting being performed under conditions determined to permit thecomposition to suppress expression of the particular allele.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene,

the contacting being performed under conditions determined to permit thecomposition to suppress transcripts of the particular allele.

In some embodiments, the present disclosure provides a method forallele-specific suppression of a transcript from a target gene for whicha plurality of alleles exist within a population, each of which containsa specific nucleotide characteristic sequence element that defines theallele relative to other alleles of the same target gene, the methodcomprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene,

the contacting being performed under conditions determined to permit thecomposition to suppress expression of the particular allele.

As described herein, in some embodiments, in a provided methodcontacting is performed under conditions determined to permit acomposition to suppress transcripts of a particular allele. In someembodiments, contacting is performed under conditions determined topermit a composition to suppress expression of a particular allele.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than when the composition is absent. Insome embodiments, suppression of transcripts of a particular allele isat a level that is at least 1.1 fold relative to when the composition isabsent, in that transcripts from the particular allele are detected inamounts that are at least 1.1 fold lower when the composition is presentrelative to when it is absent. In some embodiments, a level is at least1.2 fold. In some embodiments, a level is at least 1.3 fold. In someembodiments, a level is at least 1.4 fold. In some embodiments, a levelis at least 1.5 fold. In some embodiments, a level is at least 1.6 fold.In some embodiments, a level is at least 1.7 fold. In some embodiments,a level is at least 1.8 fold. In some embodiments, a level is at least1.9 fold. In some embodiments, a level is at least 2 fold. In someembodiments, a level is at least 3 fold. In some embodiments, a level isat least 4 fold. In some embodiments, a level is at least 5 fold. Insome embodiments, a level is at least 6 fold. In some embodiments, alevel is at least 7 fold. In some embodiments, a level is at least 8fold. In some embodiments, a level is at least 9 fold. In someembodiments, a level is at least 10 fold. In some embodiments, a levelis at least 11 fold. In some embodiments, a level is at least 12 fold.In some embodiments, a level is at least 13 fold. In some embodiments, alevel is at least 14 fold. In some embodiments, a level is at least 15fold. In some embodiments, a level is at least 20 fold. In someembodiments, a level is at least 30 fold. In some embodiments, a levelis at least 40 fold. In some embodiments, a level is at least 50 fold.In some embodiments, a level is at least 75 fold. In some embodiments, alevel is at least 100 fold. In some embodiments, a level is at least 150fold. In some embodiments, a level is at least 200 fold. In someembodiments, a level is at least 300 fold. In some embodiments, a levelis at least 400 fold. In some embodiments, a level is at least 500 fold.In some embodiments, a level is at least 750 fold. In some embodiments,a level is at least 1000 fold. In some embodiments, a level is at least5000 fold.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments,suppression of transcripts of a particular allele is at a level that isat least 1.1 fold greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments, alevel is at least 1.2 fold. In some embodiments, a level is at least 1.3fold. In some embodiments, a level is at least 1.4 fold. In someembodiments, a level is at least 1.5 fold. In some embodiments, a levelis at least 1.6 fold. In some embodiments, a level is at least 1.7 fold.In some embodiments, a level is at least 1.8 fold. In some embodiments,a level is at least 1.9 fold. In some embodiments, a level is at least 2fold. In some embodiments, a level is at least 3 fold. In someembodiments, a level is at least 4 fold. In some embodiments, a level isat least 5 fold. In some embodiments, a level is at least 6 fold. Insome embodiments, a level is at least 7 fold. In some embodiments, alevel is at least 8 fold. In some embodiments, a level is at least 9fold. In some embodiments, a level is at least 10 fold. In someembodiments, a level is at least 11 fold. In some embodiments, a levelis at least 12 fold. In some embodiments, a level is at least 13 fold.In some embodiments, a level is at least 14 fold. In some embodiments, alevel is at least 15 fold. In some embodiments, a level is at least 20fold. In some embodiments, a level is at least 30 fold. In someembodiments, a level is at least 40 fold. In some embodiments, a levelis at least 50 fold. In some embodiments, a level is at least 75 fold.In some embodiments, a level is at least 100 fold. In some embodiments,a level is at least 150 fold. In some embodiments, a level is at least200 fold. In some embodiments, a level is at least 300 fold. In someembodiments, a level is at least 400 fold. In some embodiments, a levelis at least 500 fold. In some embodiments, a level is at least 750 fold.In some embodiments, a level is at least 1000 fold. In some embodiments,a level is at least 5000 fold.

In some embodiments, suppression of transcripts of a particular alleleis at a level that is greater than when the composition is absent, andat a level that is greater than a level of suppression observed foranother allele of the same nucleic acid sequence. In some embodiments,suppression of transcripts of a particular allele is at a level that isat least 1.1 fold relative to when the composition is absent, and atleast 1.1 fold greater than a level of suppression observed for anotherallele of the same nucleic acid sequence. In some embodiments, each foldis independently as described above.

In some embodiments, a system is a composition comprising a transcript.In some embodiments, a system is a composition comprising transcriptsfrom different alleles. In some embodiments, a system can be in vivo orin vitro, and in either way can comprise one or more cells, tissues,organs or organisms. In some embodiments, a system comprises one or morecells. In some embodiments, a system comprises one or more tissues. Insome embodiments, a system comprises one or more organs. In someembodiments, a system comprises one or more organisms. In someembodiments, a system is a subject.

In some embodiments, suppression of a transcript, or suppression ofexpression of an allele from which a transcript is transcribed, can bemeasured in in vitro assay. In some embodiments, a sequence from atranscript and comprising a specific nucleotide characteristic sequenceelement is used in assays instead of the full-length transcript. In someembodiments, an assay is a biochemical assay. In some embodiments, anassay is a biochemical assay wherein a nucleic acid polymer, forexample, a transcript or a sequence from a transcript and comprising aspecific nucleotide characteristic sequence element, is tested forcleavage by an enzyme in the presence of a chirally controlledoligonucleotide composition.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is administered to a subject. In some embodiments, a subjectis an animal. In some embodiments, a subject is a plant. In someembodiments, a subject is a human.

In some embodiments, for allele-specific suppression of transcripts froma particular allele, transcripts are cleaved at a site near a sequencedifference, for example a mutation, within a specific nucleotidecharacteristic sequence element, which sequence differencedifferentiates transcripts from a particular allele from transcriptsfrom the other alleles. In some embodiments, transcripts are selectivelycleaved at a site near such a sequence difference. In some embodiments,transcripts are cleaved at a higher percentage at a site near such asequence difference that when a chirally uncontrolled oligonucleotidecomposition is used. In some embodiments, transcripts are cleaved at thesite of a sequence difference. In some embodiments, transcripts arecleaved only at the site of a sequence difference within a specificnucleotide characteristic sequence element. In some embodiments,transcripts are cleaved at a site within 5 base pairs downstream orupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 4 base pairs downstream or upstream a sequencedifference. In some embodiments, transcripts are cleaved at a sitewithin 3 base pairs downstream or upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 2 base pairsdownstream or upstream a sequence difference. In some embodiments,transcripts are cleaved at a site within 1 base pair downstream orupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 5 base pairs downstream a sequence difference.In some embodiments, transcripts are cleaved at a site within 4 basepairs downstream a sequence difference. In some embodiments, transcriptsare cleaved at a site within 3 base pairs downstream a sequencedifference. In some embodiments, transcripts are cleaved at a sitewithin 2 base pairs downstream a sequence difference. In someembodiments, transcripts are cleaved at a site within 1 base pairdownstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 5 base pairs upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 4 base pairsupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 3 base pairs upstream a sequence difference. Insome embodiments, transcripts are cleaved at a site within 2 base pairsupstream a sequence difference. In some embodiments, transcripts arecleaved at a site within 1 base pair upstream a sequence difference.Such precise control of cleavage patterns, and the resulting highlyselective suppression of transcripts from a particular allele, would notbe possible without chirally controlled oligonucleotide compositions andmethods thereof provided by Applicant in this disclosure.

In some embodiments, the present disclosure provides methods fortreating a subject, or preventing a disease in a subject, byspecifically suppress transcripts from a particular allele, for example,an allele that causes or may cause a disease. In some embodiments, thepresent disclosure provides methods for treating a subject sufferingfrom a disease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that causes orcontributes to the disease is selectively suppressed. In someembodiments, the present disclosure provides methods for treating asubject suffering from a disease, comprising administering to thesubject a pharmaceutical composition comprising a chirally controlledoligonucleotide composition, wherein transcripts from an allele thatcauses the disease is selectively suppressed. In some embodiments, thepresent disclosure provides methods for treating a subject sufferingfrom a disease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that contributes to thedisease is selectively suppressed. In some embodiments, the presentdisclosure provides methods for treating a subject suffering from adisease, comprising administering to the subject a pharmaceuticalcomposition comprising a chirally controlled oligonucleotidecomposition, wherein transcripts from an allele that is related to thedisease is selectively suppressed. In some embodiments, the presentdisclosure provides methods for preventing a disease in a subject, byspecifically suppress transcripts from a particular allele that maycause a disease. In some embodiments, the present disclosure providesmethods for preventing a disease in a subject, by specifically suppresstranscripts from a particular allele that increases risk of a disease inthe subject. In some embodiments, a provided method comprisesadministering to the subject a pharmaceutical composition comprising achirally controlled oligonucleotide composition. In some embodiments, apharmaceutical composition further comprises a pharmaceutical carrier.

In some embodiments, a nucleotide characteristic sequence comprises amutation that defines the target sequence relative to other similarsequences. In some embodiments, a nucleotide characteristic sequencecomprises a point mutation that defines the target sequence relative toother similar sequences. In some embodiments, a nucleotidecharacteristic sequence comprises a SNP that defines the target sequencerelative to other similar sequences.

In some embodiments, the present disclosure provides a method forpreparing an oligonucleotide composition for selective suppression of atranscript of a target nucleic acid sequence, comprising providing anoligonucleotide composition comprising a predetermined level ofoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises(Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein each of m, n, t, Np is independentlyas defined and described herein;

wherein the target nucleic acid sequence comprises a characteristicsequence element that defines the target nucleic acid sequence relativeto a similar nucleic acid sequence;

wherein the common base sequence is a sequence whose DNA cleavagepattern and/or stereorandom cleavage pattern has a cleavage site withinor in the vicinity of the characteristic sequence element.

In some embodiments, the present disclosure provides a method forpreparing an oligonucleotide composition for selective suppression of atranscript of a target nucleic acid sequence, comprising providing anoligonucleotide composition comprising a predetermined level ofoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises(Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein each of m, n, t, Np is independentlyas defined and described herein;

wherein the target nucleic acid sequence comprises a characteristicsequence element that defines the target nucleic acid sequence relativeto a similar nucleic acid sequence;

wherein the common base sequence is a sequence whose DNA cleavagepattern and/or stereorandom cleavage pattern has a major cleavage sitewithin or in the vicinity of characteristic sequence element.

In some embodiments, a common pattern of backbone chiral centerscomprises (Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m),or (Sp)_(t)(Rp)_(n)(Sp)_(m) as described above. In some embodiments, acommon pattern of backbone chiral centers comprises (Sp)_(m)(Rp)_(n) asdescribed above. In some embodiments, a common pattern of backbonechiral centers comprises (Rp)_(n)(Sp)_(m) as described above. In someembodiments, a common pattern of backbone chiral centers comprises(Np)_(t)(Rp)_(n)(Sp)_(m) as described above. In some embodiments, acommon pattern of backbone chiral centers comprises(Sp)_(t)(Rp)_(n)(Sp)_(m) as described above. In some embodiments, nis 1. In some embodiments, m>2. In some embodiments, n is 1 and m>2. Insome embodiments, t>2. In some embodiments, n is 1, m>2, and t>2.

In some embodiments, oligonucleotides of the particular oligonucleotidetype have a wing-core structure. In some embodiments, oligonucleotidesof the particular oligonucleotide type have a core-wing structure. Insome embodiments, oligonucleotides of the particular oligonucleotidetype have a wing-core-wing structure. In some embodiments, each sugarmoieties in the wing regions has a sugar modification. In someembodiments, each sugar moiety in the wing regions has a2′-modification. In some embodiments, each sugar moieties in the wingregions has a 2′-modification, wherein the 2′-modification is 2′-OR¹,wherein R¹ is optionally substituted C₁₋₆ alkyl. In some embodiments,each sugar moiety in the wing regions has 2′-OMe. In some embodiments,each wing independently comprises a chiral internucleotidic linkage anda natural phosphate linkage. In some embodiments, a chiralinternucleotidic linkage is phosphorothioate. In some embodiments, for awing-core-wing structure, like in WV-1092, the 5′-wing has an Spinternucleotidic linkage at each of its 5′- and 3′-end, and phosphatelinkages in between, and 3′-wing has an Sp internucleotidic linkage atits 3′-end, and the rest of its internucleotidic linkages are phosphate.Additional embodiments for the wing and/or core, e.g., sugarmodification, stereochemistry, etc., are described in the presentdisclosure.

Common base sequences which are sequences whose DNA cleavage patternsand/or stereorandom cleavage patterns have cleavage sites within or inthe vicinity of the target nucleic acid sequence are extensivelydescribed in the present disclose. In some embodiments, a cleavage sitewithin or in the vicinity of the target nucleic acid sequence is acleavage site in the vicinity of a mutation which defines the targetsequence from its similar sequences. In some embodiments, a cleavagesite within or in the vicinity of the target nucleic acid sequence is acleavage site in the vicinity of a SNP which defines the target sequencefrom its similar sequences. In some embodiments, as described above, inthe vicinity of a mutation or a SNP is 0, 1, 2, 3, 4, 5 internucleotidiclinkages away from the mutation or SNP. Additional embodiments aredescribed above in the present disclosure.

In some embodiments, a common base sequence is a sequence whose DNAcleavage pattern and/or stereorandom cleavage pattern has a majorcleavage site within or in the vicinity of the target nucleic acidsequence. In some embodiments, a major cleavage site is defined byabsolute cleavage at that site (% of cleavage at that site over totaltarget sequence). Additional example embodiments of a major cleavagesite are described in the present disclosure. In some embodiments, asexemplified by FIG. 33, a common base sequence (P12) may be identifiedby comparing cleavage maps of different sequences complementary to thecharacteristic sequence element.

In some embodiments, the present disclosure provides a method forpreparing an oligonucleotide composition comprising oligonucleotides ofa particular sequence, which composition provides selective suppressionof a transcript of a target sequence, comprising providing a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence which is the same as the particular sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises(Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein:

each n and t is independently 1, 2, 3, 4, 5, 6, 7 or 8;

m is 2, 3, 4, 5, 6, 7 or 8, and

each Np is independent Rp or Sp.

Diseases that involves disease-causing alleles are widely known in theart, including but not limited to those described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; US patent application publication US2013/0197061; Østergaard et al., Nucleic Acids Research 2013, 41(21),9634-9650; and Jiang et al., Science 2013, 342, 111-114. In someembodiments, a disease is Huntington's disease. In some embodiments, adisease is human hypertrophic cardiomyopathy (HCM). In some embodiments,a disease is dilated cardiomyopathy. In some embodiments, adisease-causing allele is an allele of myosin heavy chains (MHC). Insome embodiments, an example disease is selected from:

Disease Target gene Target variation Familial Alzheimer's AmyloidK670N-M671L disease precursor protein (Swedish mutant) (APP) AmyloidK670N-M671L precursor protein (Swedish mutant) (APP) Amyloid V717Fprecursor protein (London mutant) (APP) Amyloid V717I precursor protein(London mutant) (APP) Preseniline 1 L392V (PSEN1) Amyotrophic lateralSuperoxide G93A sclerosis (ALS) dismutase (SOD1) Superoxide G85Rdismutase (SOD1) Slow channel Acetylcholine aS226F congenital receptor(AChR) myasthenic syndrome (SCCMS) Frontotemporal Microtubule- V337Mdementia with associated protein parkinsonism TAU (MAPT) linked tochromosome 17 (FTDP-17) Ehlers-Danlos Procollagen type III G252Vsyndrome (COL3A1) (vEDS) Sickle cell Hemoglobin-beta E6V anemia locus(HBB) Familial Transthyretin (TTR) V30M amyloidotic polyneuropathy (FAP)Fibrodysplasia Activin A receptor R206H, G356D ossificans type I (ACVR1)progressiva Activin A receptor R206H (FOP) type I (ACVR1) TumorsPhosphoinositide-3- 1633G −> A kinase, catalytic, alpha 3140A −> Gpolypeptide (PIK3CA) Spinocerebellar Ataxin-1 (ATXN1) flanking ataxiatype 1 region of (SCA1) expanded CAG repeat Machado-Joseph ATAXIN3/MJD1SNPs linked disease/ to expanded spinocerebellar CAG repeat ataxia type3 (MJD/SCA3) Spinocerebellar Ataxin-7 (ATXN7) SNP linked ataxia type 7to expanded (SCA7) CAG repeat Parkinson's Leucine-rich repeat R1441G,R1441C disease kinase 2 (LRRK2) Leucine-rich repeat G20195S kinase 2(LRRK2) alpha-synuclein A30P Huntington Huntingtin (HTT) SNPs linkeddisease to expanded CAG repeat Hypertrophic MYH7 R403Q cardiomyopathy

In some embodiments, example targets of, and diseases that can betreated by, provided chirally controlled oligonucleotide compositionsand methods, comprises:

Disease Target gene Target variation Familial Alzheimer's AmyloidK670N-M671L disease precursor protein (Swedish mutant) (APP) AmyloidK670N-M671L precursor protein (Swedish mutant) (APP) Amyloid V717Fprecursor protein (London mutant) (APP) Amyloid V717I precursor protein(London mutant) (APP) Preseniline 1 L392V (PSEN1) Amyotrophic lateralSuperoxide G93A sclerosis (ALS) dismutase (SOD1) Superoxide G85Rdismutase (SOD1) Slow channel Acetylcholine aS226F, aT254I, congenitalreceptor (AChR) aS269I myasthenic syndrome (SCCMS) FrontotemporalMicrotubule- V337M dementia with associated protein parkinsonism TAU(MAPT) linked to chromosome 17 (FTDP-17) Ehlers-Danlos Procollagen typeIII G252V syndrome (COL3A1) (vEDS) Sickle cell Hemoglobin-beta E6Vanemia locus (HBB) Familial Transthyretin (TTR) V30M amyloidoticpolyneuropathy (FAP) Fibrodysplasia Activin A receptor R206H, G356Dossificans type I (ACVR1) progressiva Activin A receptor R206H (FOP)type I (ACVR1) Tumors KRAS G12V, G12D, G13D Tumors Phosphoinositide-3-G1633A, A3140G kinase, catalytic, alpha polypeptide (PIK3CA)Spinocerebellar Ataxin-1 (ATXN1) SNPs linked ataxia type 1 to expanded(SCA1) CAG repeat Spinocerebellar Ataxin-7 (ATXN7) SNPs linked ataxiatype 7 to expanded (SCA7) CAG repeat Spinocerebellar Ataxin-3 (ATXN3)SNPs linked Ataxia Type to expanded 3 (SCA3)/Machado- CAG repeat JosephDisease Parkinson's Leucine-rich repeat R1441G, R1441C disease kinase 2(LRRK2) Leucine-rich repeat G20195S kinase 2 (LRRK2) Alpha-synuclein(SNCA) A30P, A53T, E46K Huntington's Huntingtin (HTT) SNPs linkeddisease to expanded CAG repeat Huntington's JPH3 SNPs linkeddisease-like 2 to expanded CTG repeat Friedreich's FXN SNPs linkedataxia to expanded GAA repeat Fragile X mental FMR1 SNPs linkedretardation to expanded syndrome/fragile CGG repeat X tremor ataxiasyndrome Myotonic DMPK SNPs linked Dystophy (DM1) to expanded CTG repeatMyotonic ZNF9 SNPs linked Dystophy (DM2) to expanded CTG repeatSpinal-Bulbar AR SNPs linked Muscular Atrophy to expanded CAG repeatHypertrophic MHY7 R403Q cardiomyopathy

In some embodiments, oligonucleotide compositions and technologiesdescribed herein are particularly useful in the treatment ofHuntington's disease. For example, in some embodiments, the presentdisclosure defines stereochemically controlled oligonucleotidecompositions that direct cleavage (e.g., RNAse H-mediated cleavage) ofnucleic acids associated with Huntington's disease. In some embodiments,such compositions direct preferential cleavage of a Huntington'sdisease-associated allele of a particular target sequence, relative toone or more (e.g., all non-Huntington's disease-associated) otheralleles of the sequence.

In some embodiments, a provided method for treating or preventingHuntington's disease in a subject, comprising administering to thesubject a chirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type. In some embodiments, oligonucleotidesof a particular oligonucleotide type are identical. In some embodiments,a provided composition is a chirally controlled oligonucleotidecomposition.

SNPs related to Huntington's disease are widely known in the art. Insome embodiments, a common base sequence is complementary to a nucleicacid sequence comprising a SNP related to Huntington's disease. In someembodiments, a provided composition selectively suppresses transcriptsfrom the disease-causing allele. In some embodiments, a providedcomposition selectively cleaves transcripts from the disease-causingallele. Example SNPs that can be targeted by a provided composition(target Huntingtin sites) are described herein.

In some embodiments, a target Huntingtin site is selected fromrs9993542_C, rs362310_C, rs362303_C, rs10488840_G, rs363125_C,rs363072_A, rs7694687_C, rs363064_C, rs363099_C, rs363088_A,rs34315806_C, rs2298967_T, rs362272 G, rs362275_C, rs362306_G,rs3775061_A, rs1006798_A, rs16843804_C, rs3121419_C, rs362271_G,rs362273_A, rs7659144_C, rs3129322_T, rs3121417_G, rs3095074_G,rs362296_C, rs108850_C, rs2024115_A, rs916171_C, rs7685686_A,rs6844859_T, rs4690073_G, rs2285086_A, rs362331_T, rs363092_C,rs3856973_G, rs4690072_T, rs7691627_G, rs2298969_A, rs2857936_C,rs6446723_T, rs762855_A, rs1263309_T, rs2798296G_, rs363096_T,rs10015979_G, rs11731237_T, rs363080_C, rs2798235_G and rs362307_T. Insome embodiments, a target Huntingtin site is selected fromrs34315806_C, rs362273_A, rs362331_T, rs363099_C, rs7685686_A,rs362306_G, rs363064_C, rs363075_G, rs2276881_G, rs362271_G, rs362303_C,rs362322_A, rs363088_A, rs6844859_T, rs3025838_C, rs363081_G,rs3025849_A, rs3121419_C, rs2298967_T, rs2298969_A, rs16843804_C,rs4690072_T, rs362310_C, rs3856973_G, rs2530595_C, rs2530595_T, andrs2285086_A. In some embodiments, a target Huntingtin site is selectedfrom rs34315806_C, rs362273_A, rs362331_T, rs363099_C, rs7685686_A,rs362306_G, rs363064_C, rs363075_G, rs2276881_G, rs362271_G, rs362303_C,rs362322_A, rs363088_A, rs6844859_T, rs3025838_C, rs363081_G,rs3025849_A, rs3121419_C, rs2298967_T, rs2298969_A, rs16843804_C,rs4690072_T, rs362310_C, rs3856973_G, and rs2285086_A. In someembodiments, a target Huntingtin site is selected from rs362331_T,rs7685686_A, rs6844859_T, rs2298969_A, rs4690072_T, rs2024115_A,rs3856973_G, rs2285086_A, rs363092_C, rs7691627_G, rs10015979_G,rs916171_C, rs6446723_T, rs11731237_T, rs362272_G, rs4690073_G, andrs363096_T. In some embodiments, a target Huntingtin site is selectedfrom rs362267, rs6844859, rs1065746, rs7685686, rs362331, rs362336,rs2024115, rs362275, rs362273, rs362272, rs3025805, rs3025806,rs35892913, rs363125, rs17781557, rs4690072, rs4690074, rs1557210,rs363088, rs362268, rs362308, rs362307, rs362306, rs362305, rs362304,rs362303, rs362302, rs363075, rs2530595, and rs2298969. In someembodiments, a target Huntingtin site is selected from rs362267,rs6844859, rs1065746, rs7685686, rs362331, rs362336, rs2024115,rs362275, rs362273, rs362272, rs3025805, rs3025806, rs35892913,rs363125, rs17781557, rs4690072, rs4690074, rs1557210, rs363088,rs362268, rs362308, rs362307, rs362306, rs362305, rs362304, rs362303,rs362302, rs363075 and rs2298969. In some embodiments, a targetHuntingtin site is selected from:

Frequency of Heterozygosity for 24 SNP Sites in the Huntingtin mRNALocation in mRNA Reference Percent Heterozygosity (Position, nt) NumberControls HD Patients ORF, exon 20 rs363075 G/A, 10.3% (G/G, G/A, 12.8%(G/G, (2822) 89.7%) 86.2%; A/A, 0.9%) ORF, exon 25 rs35892913 G/A, 10.3%(G/G, G/A, 13.0% (G/G, (3335) 89.7%) 86.1%; A/A, 0.9%) ORF, exon 25rs1065746 G/C, 0% (G/G, G/C, 0.9% (G/G, (3389) 100%) 99.1%) ORF, exon 25rs17781557 T/G, 12.9% (T/T, T/G, 1.9% (T/T, (3418) 87.1%) 98.1%) ORF,exon 29 rs4690074 C/T, 37.9% (C/C, C/T, 35.8% (C/C, (3946) 50.9%; T/T,11.2) 59.6%; T/T, 4.6%) ORF, exon 39 rs363125 C/A, 17.5% (C/C, C/A,11.0% (C/C, (5304) 79.0%; A/A, 3.5%) 87.2%; A/A, 1.8%) ORF, exon 44 exon44 G/A, 0% (G/G, G/A, 2.8% (G/G, (6150) 100%) 97.2%) ORF, exon 48rs362336 G/A, 38.7% (G/G, G/A, 37.4% (G/G, (6736) 49.6%; A/A, 11.7%)57.9%; A/A, 4.7%) ORF, exon 50 rs362331 T/C, 45.7% (T/T, T/C, 39.4%(T/T, (7070) 31.0%; C/C, 23.3%) 49.5%; C/C, 11.0%) ORF, exon 57 rs362273A/G, 40.3% (A/A, A/G, 35.2% (A/A, (7942) 48.2%; G/G, 11.4%) 60.2%; G/G,4.6%) ORF, exon 61 rs362272 G/A, 37.1% (G/G, G/A, 36.1% (G/G, (8501)51.7%; A/A, 11.2%) 59.3%; A/A, 4.6%) ORF, exon 65 rs3025806 A/T, 0%(C/C, A/T, 0% (C/C, (9053) 100%) 100%) ORF, exon 65 exon 65 G/A, 2.3%(G/G, G/A, 0% (G/G, (9175) 97.7%) 100%) ORF, exon 67 rs362308 T/C, 0%(T/T, T/C, 0% (T/T, (9523) 100%) 100%) 3′UTR, exon rs362307 C/T, 13.0%(C/C, C/T, 48.6% (C/C, 67 (9633) 87.0%) 49.5%; T/T, 1.9%) 3′UTR, exonrs362306 G/A, 36.0% (G/G, G/A, 35.8% (G/G, 67 (9888) 52.6%; A/A, 11.4%)59.6%; A/A, 4.6%) 3′UTR, exon rs362268 C/G, 36.8% (C/C, C/G, 35.8% (C/C,67 (9936) 50.0%; G/G 13.2%) 59.6%; G/G, 4.6%) 3′UTR, exon rs362305 C/G,20.2% (C/C, C/G, 11.9% (C/C, 67 (9948) 78.1%; G/G 1.8%) 85.3%; G/G,2.8%) 3′UTR, exon rs362304 C/A, 22.8% (C/C, C/A, 11.9% (C/C, 67 (10060)73.7%; A/A, 3.5%) 85.3%; AA, 2.8%) 3′UTR, exon rs362303 C/T, 18.4% (C/C,C/A, 11.9% (C/C, 67 (10095) 79.8%; T/T, 1.8%) 85.3%; T/T, 2.8%) 3′UTR,exon rs1557210 C/T, 0% (C/C, C/T, 0% (C/C, 67 (10704) 100%) 100%) 3′UTR,exon rs362302 C/T, 4.3% (C/C, C/T, 0% (C/C, 67 (10708) 95.7%) 100%)3′UTR, exon rs3025805 G/T, 0% (G/G, G/T, 0% (G/G, 67 (10796) 100%) 100%)3′UTR, exon rs362267 C/T, 36.2% (C/C, C/T, 35.5% (C/C, 67 (11006) 52.6%;T/T, 11.2%) 59.8%; T/T, 4.7%)

In some embodiments, a chirally controlled oligonucleotide compositiontargets two or more sites. In some embodiments, targeted two or moresites are selected from sited listed herein. In some embodiments, atargeted SNP is rs362307, rs7685686, rs362268, rs2530595, rs362331, orrs362306. In some embodiments, a targeted SNP is rs362307, rs7685686,rs362268 or rs362306. In some embodiments, a targeted SNP is rs362307.In some embodiments, a targeted SNP is rs7685686. In some embodiments, atargeted SNP is not rs7685686. In some embodiments, a targeted SNP isrs362268. In some embodiments, a targeted SNP is rs362306.

In some embodiments, a chirally controlled oligonucleotide compositionis able to differentiate between two alleles of a particular SNP.

A chirally controlled oligonucleotide compositions of both WVE120101 andWV-1092 were able to differentiate between wt and mutant versions of SNPrs362307, which differ by one nt; both WVE120101 and WV-1092 chirallycontrolled oligonucleotide compositions significantly knocked down themutant allele but not the wt, while the stereorandom oligonucleotidecomposition, WV-1497, was not able to significantly differentiatebetween the wt and mutant alleles (see FIG. 39D).

A chirally controlled oligonucleotide composition of WV-2595 was alsoable to differentiate between the C and T alleles at SNP rs2530595,which also differ at only the one nt. A chirally controlled WV-2595oligonucleotide composition significantly knocked down the T allele butnot the C allele, unlike the stereorandom oligonucleotide composition ofWV-2611, which was not able to significantly differentiate the alleles(see FIG. 39F).

A chirally controlled oligonucleotide composition of WV-2603 was able todifferentiate between the C and T alleles of SNP rs362331, which alsodiffer at only the one nt. A chirally controlled WV-2603 oligonucleotidecomposition significantly knocked down the T allele but not the Callele, unlike the stereorandom oligonucleotide composition of WV-2619,which was not able to significantly differentiate between the alleles(see FIGS. 39A, 39B, 39C and 39E).

In some embodiments, a provided composition for treating Huntington'sdisease is selected from Tables N1, N2, N3 or N4. In some embodiments, aprovided composition for treating Huntington's disease is selected fromTable N1. In some embodiments, a provided composition for treatingHuntington's disease is selected from Table N2. In some embodiments, aprovided composition for treating Huntington's disease is selected fromTable N3. In some embodiments, a provided composition for treatingHuntington's disease is selected from Table N4. In some embodiments, aprovided composition for treating Huntington's disease is selected fromTables N1A, N2A, N3A or N4A. In some embodiments, a provided compositionfor treating Huntington's disease is selected from Table N1A. In someembodiments, a provided composition for treating Huntington's disease isselected from Table N2A. In some embodiments, a provided composition fortreating Huntington's disease is selected from Table N3A. In someembodiments, a provided composition for treating Huntington's disease isselected from Table N4A. In some embodiments, a provided composition isWV-1092. In some embodiments, a provided composition is selected from:an oligonucleotide having a sequence of WVE120101; WV-2603; WV-2595;WV-1510; WV-2378; and WV-2380; each of these was constructed and foundto be very effective, for example, as demonstrated in vitro in the dualluciferase reporter assay. In some embodiments, a provided compositionis a chirally controlled oligonucleotide composition of WVE120101,WV-2603, WV-2595, WV-1510, WV-2378, or WV-2380. In some embodiments, aprovided composition is a chirally controlled oligonucleotidecomposition of WV-937. In some embodiments, a provided composition is achirally controlled oligonucleotide composition of WV-1087. In someembodiments, a provided composition is a chirally controlledoligonucleotide composition of WV-1090. In some embodiments, a providedcomposition is a chirally controlled oligonucleotide composition ofWV-1091. In some embodiments, a provided composition is a chirallycontrolled oligonucleotide composition of WV-1092. In some embodiments,a provided composition is a chirally controlled oligonucleotidecomposition of WV-1510. In some embodiments, a provided composition is achirally controlled oligonucleotide composition of WV-2378. In someembodiments, a provided composition is a chirally controlledoligonucleotide composition of WV-2380. In some embodiments, a providedcomposition is a chirally controlled oligonucleotide composition ofWV-2595. In some embodiments, a provided composition is a chirallycontrolled oligonucleotide composition of WV-2603. In some embodiments,provided oligonucleotides comprise base sequence, pattern of backbonelinkages, pattern or backbone chiral centers, and/or pattern of chemicalmodifications (e.g., base modifications, sugar modifications, etc.) ofany oligonucleotide disclosed herein.

In some embodiments, a provided composition is not a composition ofONT-451, ONT-452 or ONT-450. In some embodiments, a provided compositionis not a composition of ONT-451. In some embodiments, a providedcomposition is not a composition of ONT-452. In some embodiments, aprovided composition is not a composition of ONT-450. In someembodiments, a composition does not contain a pre-determined level ofONT-451 or ONT-452. In some embodiments, a composition does not containa pre-determined level of ONT-451. In some embodiments, a compositiondoes not contain a pre-determined level of ONT-452. In some embodiments,an oligonucleotide type is not ONT-451 or ONT-452. In some embodiments,an oligonucleotide type is not ONT-451. In some embodiments, anoligonucleotide type is not ONT-452. In some embodiments, a compositionis not a chirally controlled oligonucleotide composition of ONT-451 orONT-452. In some embodiments, a composition is not a chirally controlledoligonucleotide composition of ONT-451. In some embodiments, acomposition is not a chirally controlled oligonucleotide composition ofONT-452.

In some embodiments, a provided method ameliorates a symptom ofHuntington's disease. In some embodiments, a provided method slows onsetof Huntington's disease. In some embodiments, a provided method slowsprogression of Huntington's disease. In some embodiments, a providedmethod stops progression of Huntington's disease. In some embodiments, aprovided method cures Huntington's disease according to a clinicalstandard.

In some embodiments, the present disclosure provides methods foridentifying patients for a given oligonucleotide composition. In someembodiments, the present disclosure provides methods for patientstratification. In some embodiments, a provided method comprisesidentifying a mutation and/or SNP associated with a disease-causingallele. For example, in some embodiments, a provided method comprisesidentifying in a subject a SNP associated with expanded CAG repeats thatare associated with or causing Huntington's disease. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 35 CAG repeats in Huntingtin. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 36 CAG repeats in Huntingtin. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 37 CAG repeats in Huntingtin. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 38 CAG repeats in Huntingtin. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 39 CAG repeats in Huntingtin. In someembodiments, a provided method comprises identifying in a subject a SNPassociated with more than 40 CAG repeats in Huntingtin.

In some embodiments, a subject has a SNP in the subject's Huntingtingene. In some embodiments, a subject has a SNP, wherein one allele ismutant Huntingtin associated with expanded CAG repeats. In someembodiments, a subject has a SNP as described herein. In someembodiments, a subject has a SNP selected from rs362307, rs7685686,rs362268, rs2530595, rs362331, or rs362306. In some embodiments, asubject has a SNP selected from rs362307, rs7685686, rs362268, orrs362306. In some embodiments, a subject has a SNP selected fromrs362307. In some embodiments, a subject has a SNP selected fromrs7685686. In some embodiments, a subject has a SNP selected fromrs362268. In some embodiments, a subject has a SNP selected fromrs362306.

In some embodiments, oligonucleotides of a provided composition have asequence complementary to a sequence comprising a SNP from thedisease-causing allele (mutant), and the composition selectivelysuppresses expression from the diseasing-causing allele. In someembodiments, a SNP is rs362307, rs7685686, rs362268, rs2530595,rs362331, or rs362306. In some embodiments, a SNP is rs362307,rs7685686, rs362268, or rs362306. In some embodiments, a SNP isrs362307. In some embodiments, a SNP is rs7685686. In some embodiments,a SNP is rs362268. In some embodiments, a SNP is rs362306. In someembodiments, a SNP is rs2530595. In some embodiments, a SNP is rs362331.

As understood by a person having ordinary skill in the art, variousmethods may be used to monitor a treatment process. In some embodiments,mutant HTT (mHTT) may be assessed from cerebrospinal fluid (Wild et al.,Quantification of mutant Huntingtin protein in cerebrospinal fluid fromHuntington's disease patients, J Clin Invest. 2015; 125 (5): 1979-86),and may be used to monitor a treatment. In some embodiments, thisapproach may be used to determined and/or optimize a regimen, monitorpharmacodynamic endpoints, and/or determine dosage and frequency foradministration, etc.

It is understood by a person having ordinary skill in the art thatprovided methods apply to any similar targets containing a mismatch. Insome embodiments, a mismatch is between a maternal and paternal gene.Additional example targets for suppression and/or knockdown, includingallele-specific suppression and/or knockdown, can be any geneticabnormalities, e.g., mutations, related to any diseases. In someembodiments, a target, or a set of targets, is selected from geneticdeterminants of diseases, e.g., as disclosed in Xiong, et al., The humansplicing code reveals new insights into the genetic determinants ofdisease. Science Vol. 347 no. 6218 DOI: 10.1126/science.1254806. In someembodiments, a mismatch is between a mutant and a wild type.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods are used to selectively suppressoligonucleotides with a mutation in a disease. In some embodiments, adisease is cancer. In some embodiments, provided chirally controlledoligonucleotide compositions and methods are used to selectivelysuppress transcripts with mutations in cancer. In some embodiments,provided chirally controlled oligonucleotide compositions and methodsare used to suppress transcripts of KRAS. Example target KRAS sitescomprises G12V=GGU->GUU Position 227 G->U, G12D=GGU->GAU Position 227G->A and G13D=GGC->GAC Position 230 G->A.

In some embodiments, provided chirally controlled oligonucleotidecompositions and methods provide allele-specific suppression of atranscript in an organism. In some embodiments, an organism comprises atarget gene for which two or more alleles exist. For example, a subjecthas a wild type gene in its normal tissues, while the same gene ismutated in diseased tissues such as in a tumor. In some embodiments, thepresent disclosure provides chirally controlled oligonucleotidecompositions and methods that selectively suppress one allele, forexample, one with a mutation or SNP. In some embodiments, the presentdisclosure provides treatment with higher efficacy and/or low toxicity,and/or other benefits as described in the application.

In some embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of only one oligonucleotidetype. In some embodiments, provided chirally controlled oligonucleotidecompositions has oligonucleotides of only one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of two or more oligonucleotidetypes. In some embodiments, using such compositions, provided methodscan target more than one target. In some embodiments, a chirallycontrolled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more targets. In some embodiments,a chirally controlled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more mismatches. In someembodiments, a single oligonucleotide type targets two or more targets,e.g., mutations. In some embodiments, a target region ofoligonucleotides of one oligonucleotide type comprises two or more“target sites” such as two mutations or SNPs.

In some embodiments, oligonucleotides in a provided chirally controlledoligonucleotide composition optionally comprise modified bases orsugars. In some embodiments, a provided chirally controlledoligonucleotide composition does not have any modified bases or sugars.In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified bases. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise modified bases and sugars. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified base. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified sugar. Modified bases and sugars foroligonucleotides are widely known in the art, including but not limitedin those described in the present disclosure. In some embodiments, amodified base is 5-mC. In some embodiments, a modified sugar is a2′-modified sugar. Suitable 2′-modification of oligonucleotide sugarsare widely known by a person having ordinary skill in the art. In someembodiments, 2′-modifications include but are not limited to 2′-OR¹,wherein R¹ is not hydrogen. In some embodiments, a 2′-modification is2′-OR, wherein R¹ is optionally substituted C₁₋₆ aliphatic. In someembodiments, a 2′-modification is 2′-MOE. In some embodiments, amodification is 2′-halogen. In some embodiments, a modification is 2′-F.In some embodiments, modified bases or sugars may further enhanceactivity, stability and/or selectivity of a chirally controlledoligonucleotide composition, whose common pattern of backbone chiralcenters provides unexpected activity, stability and/or selectivity.

In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified sugars. In some embodiments, aprovided chirally controlled oligonucleotide composition does not haveany 2′-modified sugars. In some embodiments, the present disclosuresurprisingly found that by using chirally controlled oligonucleotidecompositions, modified sugars are not needed for stability, activity,and/or control of cleavage patterns. Furthermore, in some embodiments,the present disclosure surprisingly found that chirally controlledoligonucleotide compositions of oligonucleotides without modified sugarsdeliver better properties in terms of stability, activity, turn-overand/or control of cleavage patterns. For example, in some embodiments,it is surprisingly found that chirally controlled oligonucleotidecompositions of oligonucleotides having no modified sugars dissociatesmuch faster from cleavage products and provide significantly increasedturn-over than compositions of oligonucleotides with modified sugars.

In some embodiments, oligonucleotides of provided chirally controlledoligonucleotide compositions useful for provided methods have structuresas extensively described in the present disclosure. In some embodiments,an oligonucleotide has a wing-core-wing structure as described. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises (Sp)_(m)Rp asdescribed. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)₂Rp. In some embodiments, the common pattern of backbonechiral centers of a provided chirally controlled oligonucleotidecomposition comprises (Sp)_(m)(Rp)_(n) as described. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises(Rp)_(n)(Sp)_(m) as described. In some embodiments, the common patternof backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises Rp(Sp)_(m) as described. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises Rp(Sp)₂. Insome embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)_(m)(Rp)_(n)(Sp)_(t) as described. In some embodiments, the commonpattern of backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises (Sp)_(m)Rp(Sp)_(t) as described.In some embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)_(t)(Rp)_(n)(Sp)_(m) as described. In some embodiments, the commonpattern of backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises (Sp)_(t)Rp(Sp)_(m) as described.In some embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprisesSpRpSpSp. In some embodiments, the common pattern of backbone chiralcenters of a provided chirally controlled oligonucleotide compositioncomprises (Sp)₂Rp(Sp)₂. In some embodiments, the common pattern ofbackbone chiral centers of a provided chirally controlledoligonucleotide composition comprises (Sp)₃Rp(Sp)₃. In some embodiments,the common pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Sp)₄Rp(Sp)₄. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises(Sp)_(t)Rp(Sp)₅. In some embodiments, the common pattern of backbonechiral centers of a provided chirally controlled oligonucleotidecomposition comprises SpRp(Sp)₅. In some embodiments, the common patternof backbone chiral centers of a provided chirally controlledoligonucleotide composition comprises (Sp)₂Rp(Sp)₅. In some embodiments,the common pattern of backbone chiral centers of a provided chirallycontrolled oligonucleotide composition comprises (Sp)₃Rp(Sp)₅. In someembodiments, the common pattern of backbone chiral centers of a providedchirally controlled oligonucleotide composition comprises (Sp)₄Rp(Sp)₅.In some embodiments, the common pattern of backbone chiral centers of aprovided chirally controlled oligonucleotide composition comprises(Sp)₅Rp(Sp)₅. In some embodiments, a common pattern of backbone chiralcenters has only one Rp, and each of the other internucleotidic linkagesis Sp. In some embodiments, a common base length is greater than 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 32, 35, 40, 45 or 50 as described in the present disclosure. In someembodiments, a common base length is greater than 10. In someembodiments, a common base length is greater than 11. In someembodiments, a common base length is greater than 12. In someembodiments, a common base length is greater than 13. In someembodiments, a common base length is greater than 14. In someembodiments, a common base length is greater than 15. In someembodiments, a common base length is greater than 16. In someembodiments, a common base length is greater than 17. In someembodiments, a common base length is greater than 18. In someembodiments, a common base length is greater than 19. In someembodiments, a common base length is greater than 20. In someembodiments, a common base length is greater than 21. In someembodiments, a common base length is greater than 22. In someembodiments, a common base length is greater than 23. In someembodiments, a common base length is greater than 24. In someembodiments, a common base length is greater than 25. In someembodiments, a common base length is greater than 26. In someembodiments, a common base length is greater than 27. In someembodiments, a common base length is greater than 28. In someembodiments, a common base length is greater than 29. In someembodiments, a common base length is greater than 30. In someembodiments, a common base length is greater than 31. In someembodiments, a common base length is greater than 32. In someembodiments, a common base length is greater than 33. In someembodiments, a common base length is greater than 34. In someembodiments, a common base length is greater than 35.

In some embodiments, a provided chirally controlled oligonucleotidecomposition provides higher turn-over. In some embodiments, cleavageproducts from a nucleic acid polymer dissociate from oligonucleotides ofa provided chirally controlled oligonucleotide composition at a fasterrate than from oligonucleotides of a reference oligonucleotidecomposition, for example, a chirally uncontrolled oligonucleotidecomposition. In some embodiments, a provided chirally controlledoligonucleotide composition can be administered in lower unit dosage,and/or total dosage, and/or fewer doses than chirally uncontrolledoligonucleotide composition.

In some embodiments, a chirally controlled oligonucleotide compositionprovides fewer cleavage sites in the sequence of a nucleic acid polymerthat is complementary to its common base sequence or a sequence withinits common base sequence when compared to a reference oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition provides fewer cleavage sites in the sequence of a nucleicacid polymer that is complementary to its common base sequence. In someembodiments, a nucleic acid polymer is selectively cleaved at a singlesite within the sequence that is complementary to the common basesequence, or a sequence within the common base sequence, of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition provides higher cleavagepercentage at a cleavage site within the sequence that is complementaryto the common base sequence, or a sequence within the common basesequence, of the chirally controlled oligonucleotide composition. Insome embodiments, a chirally controlled oligonucleotide compositionprovides higher cleavage percentage at a cleavage site within thesequence that is complementary to the common base sequence of thechirally controlled oligonucleotide composition. In some embodiments, asite having a higher cleavage percentage is a cleavage site when areference oligonucleotide composition is used. In some embodiments, asite having a higher cleavage percentage is a cleavage site that is notpresent when a reference oligonucleotide composition is used.

It is surprisingly found that with reduced number of cleavage sites inthe complementary sequence, cleavage rate can be unexpectedly increasedand/or higher cleavage percentage can be achieved. As demonstrated inthe examples of this disclosure, provided chirally controlledoligonucleotide compositions that produce fewer cleavage sites,especially those that provide single-site cleavage, within thecomplementary sequences of target nucleic acid polymers provide muchhigher cleavage rates and much lower levels of remaining un-cleavednucleic acid polymers. Such results are in sharp contrast to generalteachings in the art in which more cleavage sites have been pursued inorder to increase the cleavage rate.

In some embodiments, a chirally controlled oligonucleotide compositionincreases cleavage rate by 1.5 fold compared to a referenceoligonucleotide composition. In some embodiments, cleavage rate isincreased by at least 2 fold. In some embodiments, cleavage rate isincreased by at least 3 fold. In some embodiments, cleavage rate isincreased by at least 4 fold. In some embodiments, cleavage rate isincreased by at least 5 fold. In some embodiments, cleavage rate isincreased by at least 6 fold. In some embodiments, cleavage rate isincreased by at least 7 fold. In some embodiments, cleavage rate isincreased by at least 8 fold. In some embodiments, cleavage rate isincreased by at least 9 fold. In some embodiments, cleavage rate isincreased by at least 10 fold. In some embodiments, cleavage rate isincreased by at least 11 fold. In some embodiments, cleavage rate isincreased by at least 12 fold. In some embodiments, cleavage rate isincreased by at least 13 fold. In some embodiments, cleavage rate isincreased by at least 14 fold. In some embodiments, cleavage rate isincreased by at least 15 fold. In some embodiments, cleavage rate isincreased by at least 20 fold. In some embodiments, cleavage rate isincreased by at least 30 fold. In some embodiments, cleavage rate isincreased by at least 40 fold. In some embodiments, cleavage rate isincreased by at least 50 fold. In some embodiments, cleavage rate isincreased by at least 60 fold. In some embodiments, cleavage rate isincreased by at least 70 fold. In some embodiments, cleavage rate isincreased by at least 80 fold. In some embodiments, cleavage rate isincreased by at least 90 fold. In some embodiments, cleavage rate isincreased by at least 100 fold. In some embodiments, cleavage rate isincreased by at least 200 fold. In some embodiments, cleavage rate isincreased by at least 300 fold. In some embodiments, cleavage rate isincreased by at least 400 fold. In some embodiments, cleavage rate isincreased by at least 500 fold. In some embodiments, cleavage rate isincreased by at least more than 500 fold.

In some embodiments, a chirally controlled oligonucleotide compositionprovides a lower level of remaining, un-cleaved target nucleic acidpolymer compared to a reference oligonucleotide composition. In someembodiments, it is 1.5 fold lower. In some embodiments, it is at least 2fold lower. In some embodiments, it is at least 3 fold lower. In someembodiments, it is at least 4 fold lower. In some embodiments, it is atleast 5 fold lower. In some embodiments, it is at least 6 fold lower. Insome embodiments, it is at least 7 fold lower. In some embodiments, itis at least 8 fold lower. In some embodiments, it is at least 9 foldlower. In some embodiments, it is at least 10 fold lower. In someembodiments, it is at least 11 fold lower. In some embodiments, it is atleast 12 fold lower. In some embodiments, it is at least 13 fold lower.In some embodiments, it is at least 14 fold lower. In some embodiments,it is at least 15 fold lower. In some embodiments, it is at least 20fold lower. In some embodiments, it is at least 30 fold lower. In someembodiments, it is at least 40 fold lower. In some embodiments, it is atleast 50 fold lower. In some embodiments, it is at least 60 fold lower.In some embodiments, it is at least 70 fold lower. In some embodiments,it is at least 80 fold lower. In some embodiments, it is at least 90fold lower. In some embodiments, it is at least 100 fold lower. In someembodiments, it is at least 200 fold lower. In some embodiments, it isat least 300 fold lower. In some embodiments, it is at least 400 foldlower. In some embodiments, it is at least 500 fold lower. In someembodiments, it is at least 1000 fold lower.

As discussed in detail herein, the present disclosure provides, amongother things, a chirally controlled oligonucleotide composition, meaningthat the composition contains a plurality of oligonucleotides of atleast one type. Each oligonucleotide molecule of a particular “type” iscomprised of preselected (e.g., predetermined) structural elements withrespect to: (1) base sequence; (2) pattern of backbone linkages; (3)pattern of backbone chiral centers; and (4) pattern of backboneP-modification moieties. In some embodiments, provided oligonucleotidecompositions contain oligonucleotides that are prepared in a singlesynthesis process. In some embodiments, provided compositions containoligonucleotides having more than one chiral configuration within asingle oligonucleotide molecule (e.g., where different residues alongthe oligonucleotide have different stereochemistry); in some suchembodiments, such oligonucleotides may be obtained in a single synthesisprocess, without the need for secondary conjugation steps to generateindividual oligonucleotide molecules with more than one chiralconfiguration.

Oligonucleotide compositions as provided herein can be used as agentsfor modulating a number of cellular processes and machineries, includingbut not limited to, transcription, translation, immune responses,epigenetics, etc. In addition, oligonucleotide compositions as providedherein can be used as reagents for research and/or diagnostic purposes.One of ordinary skill in the art will readily recognize that the presentdisclosure herein is not limited to particular use but is applicable toany situations where the use of synthetic oligonucleotides is desirable.Among other things, provided compositions are useful in a variety oftherapeutic, diagnostic, agricultural, and/or research applications.

In some embodiments, provided oligonucleotide compositions compriseoligonucleotides and/or residues thereof that include one or morestructural modifications as described in detail herein. In someembodiments, provided oligonucleotide compositions compriseoligonucleoties that contain one or more nucleic acid analogs. In someembodiments, provided oligonucleotide compositions compriseoligonucleotides that contain one or more artificial nucleic acids orresidues (e.g., a nucleotide analog), including but not limited to: apeptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino,threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleicacid (ANA), 2′-fluoroarabinose nucleic acid (FANA), cyclohexene nucleicacid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleicacid (UNA), threose nucleic acids (TNA), and/or Xeno nucleic acids(XNA), and any combination thereof.

In any of the embodiments, the disclosure is useful foroligonucleotide-based modulation of gene expression, immune response,etc. Accordingly, stereodefined, oligonucleotide compositions of thedisclosure, which contain oligonucleotides of predetermined type (i.e.,which are chirally controlled, and optionally chirally pure), can beused in lieu of conventional stereo-random or chirally impurecounterparts. In some embodiments, provided compositions show enhancedintended effects and/or reduced unwanted side effects. Certainembodiments of biological and clinical/therapeutic applications of thedisclosure are discussed explicitly below.

Various dosing regimens can be utilized to administer provided chirallycontrolled oligonucleotide compositions. In some embodiments, multipleunit doses are administered, separated by periods of time. In someembodiments, a given composition has a recommended dosing regimen, whichmay involve one or more doses. In some embodiments, a dosing regimencomprises a plurality of doses each of which are separated from oneanother by a time period of the same length; in some embodiments, adosing regimen comprises a plurality of doses and at least two differenttime periods separating individual doses. In some embodiments, all doseswithin a dosing regimen are of the same unit dose amount. In someembodiments, different doses within a dosing regimen are of differentamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount different from the first dose amount. In someembodiments, a dosing regimen comprises a first dose in a first doseamount, followed by one or more additional doses in a second (orsubsequent) dose amount that is same as or different from the first dose(or another prior dose) amount. In some embodiments, a dosing regimencomprises administering at least one unit dose for at least one day. Insome embodiments, a dosing regimen comprises administering more than onedose over a time period of at least one day, and sometimes more than oneday. In some embodiments, a dosing regimen comprises administeringmultiple doses over a time period of at least week. In some embodiments,the time period is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, adosing regimen comprises administering one dose per week for more thanone week. In some embodiments, a dosing regimen comprises administeringone dose per week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, a dosingregimen comprises administering one dose every two weeks for more thantwo week period. In some embodiments, a dosing regimen comprisesadministering one dose every two weeks over a time period of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more(e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more)weeks. In some embodiments, a dosing regimen comprises administering onedose per month for one month. In some embodiments, a dosing regimencomprises administering one dose per month for more than one month. Insome embodiments, a dosing regimen comprises administering one dose permonth for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In someembodiments, a dosing regimen comprises administering one dose per weekfor about 10 weeks. In some embodiments, a dosing regimen comprisesadministering one dose per week for about 20 weeks. In some embodiments,a dosing regimen comprises administering one dose per week for about 30weeks. In some embodiments, a dosing regimen comprises administering onedose per week for 26 weeks. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that differs from that utilized for a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence,and/or of a different chirally controlled oligonucleotide composition ofthe same sequence. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that is reduced as compared with that of a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence inthat it achieves a lower level of total exposure over a given unit oftime, involves one or more lower unit doses, and/or includes a smallernumber of doses over a given unit of time. In some embodiments, achirally controlled oligonucleotide composition is administeredaccording to a dosing regimen that extends for a longer period of timethan does that of a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of the same sequence Without wishing to belimited by theory, Applicant notes that in some embodiments, the shorterdosing regimen, and/or longer time periods between doses, may be due tothe improved stability, bioavailability, and/or efficacy of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition has a longer dosing regimencompared to the corresponding chirally uncontrolled oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition has a shorter time period between at least two dosescompared to the corresponding chirally uncontrolled oligonucleotidecomposition. Without wishing to be limited by theory, Applicant notesthat in some embodiments longer dosing regimen, and/or shorter timeperiods between doses, may be due to the improved safety of a chirallycontrolled oligonucleotide composition.

A single dose can contain various amounts of a type of chirallycontrolled oligonucleotide, as desired suitable by the application. Insome embodiments, a single dose contains about 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ormore) mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 1 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 5 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 10 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 15 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 20 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 50 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 100 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 150 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 200 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a single dose containsabout 250 mg of a type of chirally controlled oligonucleotide. In someembodiments, a single dose contains about 300 mg of a type of chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a lower amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide. Insome embodiments, a chirally controlled oligonucleotide is administeredat a lower amount in a single dose, and/or in total dose, than achirally uncontrolled oligonucleotide due to improved efficacy. In someembodiments, a chirally controlled oligonucleotide is administered at ahigher amount in a single dose, and/or in total dose, than a chirallyuncontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a higher amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide dueto improved safety.

Biologically Active Oligonucleotides

A provided oligonucleotide composition as used herein may comprisesingle stranded and/or multiply stranded oligonucleotides. In someembodiments, single-stranded oligonucleotides contain self-complementaryportions that may hybridize under relevant conditions so that, as used,even single-stranded oligonucleotides may have at least partiallydouble-stranded character. In some embodiments, an oligonucleotideincluded in a provided composition is single-stranded, double-stranded,or triple-stranded. In some embodiments, an oligonucleotide included ina provided composition comprises a single-stranded portion and amultiple-stranded portion within the oligonucleotide. In someembodiments, as noted above, individual single-stranded oligonucleotidescan have double-stranded regions and single-stranded regions.

In some embodiments, provided compositions include one or moreoligonucleotides fully or partially complementary to strand of:structural genes, genes control and/or termination regions, and/orself-replicating systems such as viral or plasmid DNA. In someembodiments, provided compositions include one or more oligonucleotidesthat are or act as siRNAs or other RNA interference reagents (RNAiagents or iRNA agents), shRNA, antisense oligonucleotides, self-cleavingRNAs, ribozymes, fragment thereof and/or variants thereof (such asPeptidyl transferase 23S rRNA, RNase P, Group I and Group II introns,GIR1 branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerheadribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmSribozymes, CoTC ribozyme, etc.), microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, Ul adaptors, triplex-formingoligonucleotides, RNA activators, long non-coding RNAs, short non-codingRNAs (e.g., piRNAs), immunomodulatory oligonucleotides (such asimmunostimulatory oligonucleotides, immunoinhibitory oligonucleotides),GNA, LNA, ENA, PNA, TNA, HNA, TNA, XNA, HeNA, CeNA, morpholinos,G-quadruplex (RNA and DNA), antiviral oligonucleotides, and decoyoligonucleotides.

In some embodiments, provided compositions include one or more hybrid(e.g., chimeric) oligonucleotides. In the context of the presentdisclosure, the term “hybrid” broadly refers to mixed structuralcomponents of oligonucleotides. Hybrid oligonucleotides may refer to,for example, (1) an oligonucleotide molecule having mixed classes ofnucleotides, e.g., part DNA and part RNA within the single molecule(e.g., DNA-RNA); (2) complementary pairs of nucleic acids of differentclasses, such that DNA:RNA base pairing occurs either intramolecularlyor intermolecularly; or both; (3) an oligonucleotide with two or morekinds of the backbone or internucleotide linkages.

In some embodiments, provided compositions include one or moreoligonucleotide that comprises more than one classes of nucleic acidresidues within a single molecule. For example, in any of theembodiments described herein, an oligonucleotide may comprise a DNAportion and an RNA portion. In some embodiments, an oligonucleotide maycomprise a unmodified portion and modified portion.

Provided oligonucleotide compositions can include oligonucleotidescontaining any of a variety of modifications, for example as describedherein. In some embodiments, particular modifications are selected, forexample, in light of intended use. In some embodiments, it is desirableto modify one or both strands of a double-stranded oligonucleotide (or adouble-stranded portion of a single-stranded oligonucleotide). In someembodiments, the two strands (or portions) include differentmodifications. In some embodiments, the two strands include the samemodifications. One of skill in the art will appreciate that the degreeand type of modifications enabled by methods of the present disclosureallow for numerous permutations of modifications to be made. Examples ofsuch modifications are described herein and are not meant to belimiting.

The phrase “antisense strand” as used herein, refers to anoligonucleotide that is substantially or 100% complementary to a targetsequence of interest. The phrase “antisense strand” includes theantisense region of both oligonucleotides that are formed from twoseparate strands, as well as unimolecular oligonucleotides that arecapable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

The phrase “sense strand” refers to an oligonucleotide that has the samenucleoside sequence, in whole or in part, as a target sequence such as amessenger RNA or a sequence of DNA. The terms “sense strand” and“passenger strand” are used interchangeably herein.

By “target sequence” is meant any nucleic acid sequence whose expressionor activity is to be modulated. The target nucleic acid can be DNA orRNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or other RNAencoded by a gene, virus, bacteria, fungus, mammal, or plant. In someembodiments, a target sequence is associated with a disease or disorder.In some embodiments, a target sequence is or comprises a portion of theHuntingtin gene. In some embodiments, a target sequence is or comprisesa portion of the Huntingtin gene comprising a SNP.

By “specifically hybridizable” and “complementary” is meant that anucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes. In reference to the nucleic molecules of the present disclosure,the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LIT pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, /. Ain. Chem. Soc. 109:3783-3785)

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or 100% complementarity meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Less than perfect complementarity refers to thesituation in which some, but not all, nucleoside units of two strandscan hydrogen bond with each other. “Substantial complementarity” refersto polynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, e.g., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.In some embodiments, non-target sequences differ from correspondingtarget sequences by at least 5 nucleotides.

When used as therapeutics, a provided oligonucleotide is administered asa pharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a therapeutically effective amount of a providedoligonucleotide comprising, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable inactiveingredient selected from pharmaceutically acceptable diluents,pharmaceutically acceptable excipients, and pharmaceutically acceptablecarriers. In another embodiment, the pharmaceutical composition isformulated for intravenous injection, oral administration, buccaladministration, inhalation, nasal administration, topicaladministration, ophthalmic administration, intrathecal administration,or otic administration. In another embodiment, the pharmaceuticalcomposition is formulated for intravenous injection, oraladministration, buccal administration, inhalation, nasal administration,topical administration, ophthalmic administration or oticadministration. In further embodiments, the pharmaceutical compositionis a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spraysolution, a suppository, a suspension, a gel, a colloid, a dispersion, asuspension, a solution, an emulsion, an ointment, a lotion, an eye drop,an ear drop, or a preparation comprising artificial cerebrospinal fluid.In further embodiments, the pharmaceutical composition is a tablet, apill, a capsule, a liquid, an inhalant, a nasal spray solution, asuppository, a suspension, a gel, a colloid, a dispersion, a suspension,a solution, an emulsion, an ointment, a lotion, an eye drop or an eardrop. In some embodiments, a pharmaceutical composition comprisescerebrospinal fluid. In some embodiments, a pharmaceutical compositioncomprises artificial cerebrospinal fluid. In some embodiments, apharmaceutical composition comprises an oligonucleotide, wherein thesequence of the oligonucleotide comprises a sequence which targets aportion of the Huntingtin gene. In some embodiments, the sequencetargets a portion of the Huntingtin gene comprising a SNP. In someembodiments, the base sequence, pattern of backbone linkages, pattern ofbackbone chiral centers, and/or pattern of sugar modifications of theoligonucleotide is or comprises the base sequence, pattern of backbonelinkages, pattern of backbone chiral centers, and/or pattern of sugarmodifications of any oligonucleotide disclosed herein, and theoligonucleotide is comprised in a pharmaceutical composition comprisingany component of a pharmaceutical composition disclosed herein. In someembodiments, the oligonucleotide targets the Huntingtin gene (as anon-limiting example, a SNP in the Huntingtin gene), and the basesequence, pattern of backbone linkages, pattern of backbone chiralcenters, and/or pattern of sugar modifications of the oligonucleotide isor comprises the base sequence, pattern of backbone linkages, pattern ofbackbone chiral centers, and/or pattern of sugar modifications of anyoligonucleotide disclosed herein, and the oligonucleotide is comprisedin a pharmaceutical composition comprising artificial cerebrospinalfluid, and the pharmaceutical composition is administered viaintrathecal administration.

Pharmaceutical Compositions

When used as therapeutics, a provided oligonucleotide or oligonucleotidecomposition described herein is administered as a pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a therapeutically effective amount of a providedoligonucleotides, or a pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In someembodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration, intrathecal administration, or otic administration. Insome embodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration or otic administration. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop, an ear drop, or a preparationcomprising artificial cerebrospinal fluid. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop or an ear drop. In some embodiments, aprovided composition comprises cerebrospinal fluid. In some embodiments,a provided composition comprises artificial cerebrospinal fluid.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising chirally controlled oligonucleotide, orcomposition thereof, in admixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the chirally controlled oligonucleotide, or compositionthereof, described above.

A variety of supramolecular nanocarriers can be used to deliver nucleicacids. Example nanocarriers include, but are not limited to liposomes,cationic polymer complexes and various polymeric. Complexation ofnucleic acids with various polycations is another approach forintracellular delivery; this includes use of PEGlyated polycations,polyethyleneamine (PEI) complexes, cationic block co-polymers, anddendrimers. Several cationic nanocarriers, including PEI andpolyamidoamine dendrimers help to release contents from endosomes. Otherapproaches include use of polymeric nanoparticles, polymer micelles,quantum dots and lipoplexes.

Additional nucleic acid delivery strategies are known in addition to theexample delivery strategies described herein.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington, The Science andPractice of Pharmacy, (20th ed. 2000).

Provided oligonucleotides, and compositions thereof, are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100mg, from about 1 to about 50 mg per day, and from about 5 to about 100mg per day are examples of dosages that may be used. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington, The Science and Practice of Pharmacy (20th ed. 2000).Preferred pharmaceutically acceptable salts include, for example,acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide,hydrochloride, maleate, mesylate, napsylate, pamoate (embonate),phosphate, salicylate, succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington, The Science and Practice of Pharmacy (20th ed. 2000).Suitable routes may include oral, buccal, by inhalation spray,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articullar, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections or other modes ofdelivery.

In some embodiments of a method or composition of the presentdisclosure, a composition comprising an oligonucleotide is administeredvia intrathecal administration. In some embodiments of a method orcomposition of the present disclosure, a composition comprising anoligonucleotide comprises artificial cerebrospinal fluid and isadministered via intrathecal administration. In some embodiments of amethod or composition of the present disclosure, a compositioncomprising an oligonucleotide comprises one or more components ofartificial cerebrospinal fluid (for example, NaCl, NaHCO₃, KCl, NaH₂PO₄,MgCl₂ and glucose) and is administered via intrathecal administration.In some embodiments of a method or composition of the presentdisclosure, a composition comprising an oligonucleotide comprises one ormore components of artificial cerebrospinal fluid (for example, NaCl,NaHCO₃, KCl, NaH₂PO₄, MgCl₂ and glucose) and is administered viaintrathecal administration, wherein the sequence of the oligonucleotidecomprises a sequence which targets a portion of the Huntingtin gene. Insome embodiments of a method or composition of the present disclosure, acomposition comprising an oligonucleotide comprises two or morecomponents of artificial cerebrospinal fluid (for example, NaCl, NaHCO₃,KCl, NaH₂PO₄, MgCl₂ and glucose) and is administered via intrathecaladministration, wherein the sequence of the oligonucleotide comprises asequence which targets a portion of the Huntingtin gene. In someembodiments of a method or composition of the present disclosure, acomposition comprising an oligonucleotide comprises three or morecomponents of artificial cerebrospinal fluid (for example, NaCl, NaHCO₃,KCl, NaH₂PO₄, MgCl₂ and glucose) and is administered via intrathecaladministration, wherein the sequence of the oligonucleotide comprises asequence which targets a portion of the Huntingtin gene. In someembodiments, provided oligonucleotides comprise base sequence, patternof backbone linkages, pattern or backbone chiral centers, and/or patternof chemical modifications (e.g., base modifications, sugarmodifications, etc.) of any oligonucleotide disclosed herein. In someembodiments, the sequence targets a portion of the Huntingtin genecomprising a SNP.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection.

The compounds can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the disclosure tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject(e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

In certain embodiments, oligonucleotides and compositions are deliveredto the CNS. In certain embodiments, oligonucleotides and compositionsare delivered to the cerebrospinal fluid. In certain embodiments,oligonucleotides and compositions are administered to the brainparenchyma. In certain embodiments, oligonucleotides and compositionsare delivered to an animal/subject by intrathecal administration, orintracerebroventricular administration. Broad distribution ofoligonucleotides and compositions, described herein, within the centralnervous system may be achieved with intraparenchymal administration,intrathecal administration, or intracerebroventricular administration.

In certain embodiments, parenteral administration is by injection, by,e.g., a syringe, a pump, etc. In certain embodiments, the injection is abolus injection. In certain embodiments, the injection is administereddirectly to a tissue, such as striatum, caudate, cortex, hippocampus andcerebellum.

In certain embodiments, methods of specifically localizing apharmaceutical agent, such as by bolus injection, decreases medianeffective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or50. In certain embodiments, the pharmaceutical agent in an antisensecompound as further described herein. In certain embodiments, thetargeted tissue is brain tissue. In certain embodiments the targetedtissue is striatal tissue. In certain embodiments, decreasing EC50 isdesirable because it reduces the dose required to achieve apharmacological result in a patient in need thereof.

In certain embodiments, an antisense oligonucleotide is delivered byinjection or infusion once every month, every two months, every 90 days,every 3 months, every 6 months, twice a year or once a year.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with oligonucleotides of this disclosure. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the oligonucleotides of this disclosure to treatproliferative diseases and cancer. Examples of known chemotherapeuticagents include, but are not limited to, adriamycin, dexamethasone,vincristine, cyclophosphamide, fluorouracil, topotecan, taxol,interferons, and platinum derivatives.

The function and advantage of these and other embodiments of the presentdisclosure will be more fully understood from the examples describedbelow. The following examples are intended to illustrate the benefits ofthe present disclosure, but do not exemplify the full scope of thedisclosure.

Lipids

In some embodiments, provided oligonucleotide compositions furthercomprise one or more lipids. In some embodiments, the lipids areconjugated to provided oligonucleotides in the compositions. In someembodiments, two or more same or different lipids can be conjugated toone oligonucleotide, through either the same or differently chemistryand/or locations. In some embodiments, a composition can comprise anoligonucleotide disclosed herein (as non-limiting examples, a chirallycontrolled oligonucleotide composition, or a chirally controlledoligonucleotide composition wherein the sequence of the oligonucleotidecomprises, consists of or is the sequence of any oligonucleotidedisclosed herein, or a chirally controlled oligonucleotide compositionwherein the sequence of the oligonucleotide comprises, consists of or isthe sequence of any oligonucleotide disclosed in Table 8 or any otherTable herein, etc.) and a lipid. In some embodiments, a providedoligonucleotide comprises base sequence, pattern of backbone linkages,pattern or backbone chiral centers, and/or pattern of chemicalmodifications (e.g., base modifications, sugar modifications, etc.) ofany oligonucleotide disclosed herein, and is conjugated to a lipid. Insome embodiments, a provided composition comprises an oligonucleotidedisclosed herein and a lipid, wherein the lipid is conjugated to theoligonucleotide.

In some embodiments, the present disclosure provides a compositioncomprising an oligonucleotide amd a lipid. Many lipids can be utilizedin provided technologies in accordance with the present disclosure.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₈₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡—, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein:

-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₆₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein:

-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₄₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—SC(O)—, —C(O)S—, —OC(O)—, and—C(O)O—, wherein:

-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₈₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-. Insome embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₄₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

The aliphatic group of R^(LD) can be a variety of suitable length. Insome embodiments, it is C₁₀-C₈₀. In some embodiments, it is C₁₀-C₇₅. Insome embodiments, it is C₁₀-C₇₀. In some embodiments, it is C₁₀-C₆₅. Insome embodiments, it is C₁₀-C₆₀. In some embodiments, it is C₁₀-C₅₀. Insome embodiments, it is C₁₀-C₄₀. In some embodiments, it is C₁₀-C₃₅. Insome embodiments, it is C₁₀-C₃₀. In some embodiments, it is C₁₀-C₂₅. Insome embodiments, it is C₁₀-C₂₄. In some embodiments, it is C₁₀-C₂₃. Insome embodiments, it is C₁₀-C₂₂. In some embodiments, it is C₁₀-C₂₁. Insome embodiments, it is C₁₂-C₂₂. In some embodiments, it is C₁₃-C₂₂. Insome embodiments, it is C₁₄-C₂₂. In some embodiments, it is C₁₅-C₂₂. Insome embodiments, it is C₁₆-C₂₂. In some embodiments, it is C₁₇-C₂₂. Insome embodiments, it is C₁₈-C₂₂. In some embodiments, it is C₁₀-C₂₀. Insome embodiments, the lower end of the range is C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, or C₁₈. In some embodiments, the higher end of the rangeis Cis, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅,C₄₀, C₄₅, C₅₀, C₅₅, or C₆₀. In some embodiments, it is C₁₀. In someembodiments, it is C₁₁. In some embodiments, it is C₁₂. In someembodiments, it is C₁₃. In some embodiments, it is C₁₄. someembodiments, it is Cis. In some embodiments, it is C₁₆. In someembodiments, it is C₁₇. In some embodiments, it is Cis. In someembodiments, it is C₁₉. In some embodiments, it is C₂₀. In someembodiments, it is C₂₁. In some embodiments, it is C₂₂. In someembodiments, it is C₂₃. In some embodiments, it is C₂₄. In someembodiments, it is C₂₅. In some embodiments, it is C₃₀. In someembodiments, it is C₃₅. In some embodiments, it is C₄₀. In someembodiments, it is C₄₅. In some embodiments, it is C₅₀. In someembodiments, it is C₅₅. In some embodiments, it is C₆₀.

In some embodiments, a lipid comprises no more than one R^(LD) group. Insome embodiments, a lipid comprises two or more R^(LD) groups.

In some embodiments, a lipid is conjugated to a biologically activeagent, optionally through a linker, as a moiety comprising an R^(LD)group. In some embodiments, a lipid is conjugated to a biologicallyactive agent, optionally through a linker, as a moiety comprising nomore than one R^(LD) group. In some embodiments, a lipid is conjugatedto a biologically active agent, optionally through a linker, as anR^(LD) group. In some embodiments, a lipid is conjugated to abiologically active agent, optionally through a linker, as a moietycomprising two or more R^(LD) groups.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₄ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁₋₂ aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₂ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₆₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₄ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁₋₂ aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₂ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₆₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₆₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₈₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₄ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁₋₂ aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₂ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₈₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₈₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is or comprises a C₁₀ saturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₀partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₁ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₁ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₂saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₂ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₃ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₁₃ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₄ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₄ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₁₅ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₁₅ partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₆ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₆ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₇saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₇ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a Cis saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a Cis partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₉ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₉ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₀ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₂₀ partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₁ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₁ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₂₂saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₂ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₃ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₃ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₄ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₄ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₅ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₂₅ partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₆ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₆ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₂₇saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₇ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₈ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₈ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₉ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₉ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₃₀ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₃₀ partially unsaturated linear aliphatic chain.

In some embodiments, a lipid has the structure of R^(LD)—OH. In someembodiments, a lipid has the structure of R^(LD)—C(O)OH. In someembodiments, R^(LD) is

In some embodiments, a lipid is lauric acid, myristic acid, palmiticacid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid,gamma-linolenic acid, docosahexaenoic acid (DHA or cis-DHA), turbinaricacid, arachidonic acid, and dilinoleyl. In some embodiments, a lipid islauric acid, myristic acid, palmitic acid, stearic acid, oleic acid,linoleic acid, alpha-linolenic acid, gamma-linolenic acid,docosahexaenoic acid (DHA or cis-DHA), turbinaric acid, and dilinoleyl.In some embodiments, a lipid has a structure of:

In some embodiments, a lipid is, comprises or consists of any of: an atleast partially hydrophobic or amphiphilic molecule, a phospholipid, atriglyceride, a diglyceride, a monoglyceride, a fat-soluble vitamin, asterol, a fat and a wax. In some embodiments, a lipid is any of: a fattyacid, glycerolipid, glycerophospholipid, sphingolipid, sterol lipid,prenol lipid, saccharolipid, polyketide, and other molecule.

Lipids can be incorporated into provided technologies through many typesof methods in accordance with the present disclosure. In someembodiments, lipids are physically mixed with provided oligonucleotidesto form provided compositions. In some embodiments, lipids arechemically conjugated with oligonucleotides.

In some embodiments, provided compositions comprise two or more lipids.In some embodiments, provided oligonucleotides comprise two or moreconjugated lipids. In some embodiments, the two or more conjugatedlipids are the same. In some embodiments, the two or more conjugatedlipids are different. In some embodiments, provided oligonucleotidescomprise no more than one lipid. In some embodiments, oligonucleotidesof a provided composition comprise different types of conjugated lipids.In some embodiments, oligonucleotides of a provided composition comprisethe same type of lipids.

Lipids can be conjugated to oligonucleotides optionally through linkers.Various types of linkers in the art can be utilized in accordance of thepresent disclosure. In some embodiments, a linker comprise a phosphategroup, which can, for example, be used for conjugating lipids throughchemistry similar to those employed in oligonucleotide synthesis. Insome embodiments, a linker comprises an amide, ester, or ether group. Insome embodiments, a linker has the structure of -L-. In someembodiments, after conjugation to oligonucleotides, a lipid forms amoiety having the structure of -L-R^(LD), wherein each of L and R^(LD)is independently as defined and described herein.

In some embodiments, -L- comprises a bivalent aliphatic chain. In someembodiments, -L- comprises a phosphate group. In some embodiments, -L-comprises a phosphorothioate group. In some embodiments, -L- has thestructure of —C(O)NH—(CH₂)₆—OP(═O)(S⁻)—.

Lipids, optionally through linkers, can be conjugated tooligonucleotides at various suitable locations. In some embodiments,lipids are conjugated through the 5′-OH group. In some embodiments,lipids are conjugated through the 3′-OH group. In some embodiments,lipids are conjugated through one or more sugar moieties. In someembodiments, lipids are conjugated through one or more bases. In someembodiments, lipids are incorporated through one or moreinternucleotidic linkages. In some embodiments, an oligonucleotide maycontain multiple conjugated lipids which are independently conjugatedthrough its 5′-OH, 3′-OH, sugar moieties, base moieties and/orinternucleotidic linkages.

In some embodiments, a lipid is conjugated to an oligonucleotideoptionally through a linker moiety. A person having ordinary skill inthe art appreciates that various technologies can be utilized toconjugate lipids to an oligonucleotide in accordance with the presentdisclosure. For example, for lipids comprising carboxyl groups, suchlipids can be conjugated through the carboxyl groups. In someembodiments, a lipid is conjugated through a linker having the structureof -L-, wherein L is as defined and described in formula I. In someembodiments, L comprises a phosphate diester or modified phosphatediester moiety. In some embodiments, a compound formed by lipidconjugation has the structure of (R^(L)D-L-)_(x)-(oligonucleotide),wherein x is 1 or an integer greater than 1, and each of R^(LD) and L isindependently as defined and described herein. In some embodiments, xis 1. In some embodiments, x is greater than 1. In some embodiments, anoligonucleotide is an oligonucleotide. For example, in some embodiments,a conjugate has the following structures:

In some embodiments, a linker is selected from: an uncharged linker; acharged linker; a linker comprising an alkyl; a linker comprising aphosphate; a branched linker; an unbranched linker; a linker comprisingat least one cleavage group; a linker comprising at least one redoxcleavage group; a linker comprising at least one phosphate-basedcleavage group; a linker comprising at least one acid-cleavage group; alinker comprising at least one ester-based cleavage group; and a linkercomprising at least one peptide-based cleavage group.

In some embodiments, a lipid is not conjugated to an oligonucleotide.

In some embodiments, the present disclosure pertains to compositions andmethods related to a composition comprising an oligonucleotide and alipid comprising a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, wherein the lipid is conjugated to the biologicallyactive agent. In some embodiments, the present disclosure pertains tocompositions and methods related to a composition comprising anoligonucleotide and a lipid comprising a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group, wherein the lipid is conjugated to thebiologically active agent.

In some embodiments, the present disclosure pertains to compositions andmethods related to a composition comprising an oligonucleotide and alipid comprising a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, wherein the lipid is not conjugated to the biologicallyactive agent. In some embodiments, the present disclosure pertains tocompositions and methods related to a composition comprising anoligonucleotide and a lipid comprising a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group, wherein the lipid is not conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is not conjugated to the biologicallyactive agent. In some embodiments, a composition comprises anoligonucleotide and a lipid selected from: lauric acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenicacid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaricacid, and dilinoleyl, wherein the lipid is not conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is conjugated to the biologicallyactive agent. In some embodiments, a composition comprises anoligonucleotide and a lipid selected from: lauric acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenicacid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaricacid, and dilinoleyl, wherein the lipid is conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is directly conjugated to thebiologically active agent (without a linker interposed between the lipidand the biologically active agent). In some embodiments, a compositioncomprises an oligonucleotide and a lipid selected from: lauric acid,myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid,alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid(cis-DHA), turbinaric acid, and dilinoleyl, wherein the lipid isdirectly conjugated to the biologically active agent (without a linkerinterposed between the lipid and the biologically active agent).

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is indirectly conjugated to thebiologically active agent (with a linker interposed between the lipidand the biologically active agent). In some embodiments, a compositioncomprises an oligonucleotide and a lipid selected from: lauric acid,myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid,alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid(cis-DHA), turbinaric acid, and dilinoleyl, wherein the lipid isindirectly conjugated to the biologically active agent (with a linkerinterposed between the lipid and the biologically active agent).

A linker is a moiety that connects two parts of a composition; as anon-limiting example, a linker physically connects an oligonucleotide toa lipid.

Non-limiting examples of suitable linkers include: an uncharged linker;a charged linker; a linker comprising an alkyl; a linker comprising aphosphate; a branched linker; an unbranched linker; a linker comprisingat least one cleavage group; a linker comprising at least one redoxcleavage group; a linker comprising at least one phosphate-basedcleavage group; a linker comprising at least one acid-cleavage group; alinker comprising at least one ester-based cleavage group; a linkercomprising at least one peptide-based cleavage group.

In some embodiments, a linker comprises an uncharged linker or a chargedlinker.

In some embodiments, a linker comprises an alkyl.

In some embodiments, a linker comprises a phosphate. In variousembodiments, a phosphate can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon can be done. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen can be done. In variousembodiments, the linker comprising a phosphate comprises any one or moreof: a phosphorodithioate, phosphoramidate, boranophosphonoate, or acompound of formula (I):

where R³ is selected from OH, SH, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆-io aryloxy, wherein C₁₋₆ alkyl and C₆-io aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂.

In some embodiments, a linker comprises a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R₁)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In some embodiments, a linker is a branched linker. In some embodiments,a branchpoint of the branched linker may be at least trivalent, but maybe a tetravalent, pentavalent or hexavalent atom, or a group presentingsuch multiple valencies. In some embodiments, a branchpoint is —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

In one embodiment, a linker comprises at least one cleavable linkinggroup.

As a non-limiting example, a cleavable linking group can be sufficientlystable outside the cell, but which upon entry into a target cell iscleaved to release the two parts the linker is holding together. As anon-limiting example, a cleavable linking group is cleaved at least 10times or more, at least 100 times faster in the target cell or under afirst reference condition (which can, e.g., be selected to mimic orrepresent intracellular conditions) than in the blood of a subject, orunder a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum). Cleavablelinking groups are susceptible to cleavage agents, e.g., pH, redoxpotential or the presence of degradative molecules. Generally, cleavageagents are more prevalent or found at higher levels or activities insidecells than in serum or blood. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable linking group by reduction;esterases; endosomes or agents that can create an acidic environment,e.g., those that result in a pH of five or lower; enzymes that canhydrolyze or degrade an acid cleavable linking group by acting as ageneral acid, peptidases (which can be substrate specific), andphosphatases.

As a non-limiting example, a cleavable linkage group, such as adisulfide bond can be susceptible to pH. The pH of human serum is 7.4,while the average intracellular pH is slightly lower, ranging from about7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, andlysosomes have an even more acidic pH at around 5.0. Some linkers willhave a cleavable linking group that is cleaved at a desired pH, therebyreleasing the cationic lipid from the ligand inside the cell, or intothe desired compartment of the cell.

As a non-limiting example, a linker can include a cleavable linkinggroup that is cleavable by a particular enzyme. The type of cleavablelinking group incorporated into a linker can depend on the cell to betargeted. For example, liver targeting ligands can be linked to thecationic lipids through a linker that includes an ester group. Livercells are rich in esterases, and therefore the linker will be cleavedmore efficiently in liver cells than in cell types that are notesterase-rich. Other cell-types rich in esterases include cells of thelung, renal cortex, and testis.

As a non-limiting example, a linker can contain a peptide bond, whichcan be used when targeting cell types rich in peptidases, such as livercells and synoviocytes.

As a non-limiting example, suitability of a candidate cleavable linkinggroup can be evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. As a non-limiting example, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

In some embodiments, a linker comprises a redox cleavable linking group.As a non-limiting example, one class of cleavable linking groups areredox cleavable linking groups that are cleaved upon reduction oroxidation. A non-limiting example of reductively cleavable linking groupis a disulphide linking group (—S—S—). To determine if a candidatecleavable linking group is a suitable “reductively cleavable linkinggroup,” or for example is suitable for use with a particular iRNA moietyand particular targeting agent one can look to methods described herein.As a non-limiting example, a candidate can be evaluated by incubationwith dithiothreitol (DTT), or other reducing agent using reagents knowin the art, which mimic the rate of cleavage which would be observed ina cell, e.g., a target cell. The candidates can also be evaluated underconditions which are selected to mimic blood or serum conditions. As anon-limiting example, candidate compounds are cleaved by at most 10% inthe blood. As a non-limiting example, useful candidate compounds aredegraded at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

In some embodiments, a linker comprises a phosphate-based cleavablelinking groups are cleaved by agents that degrade or hydrolyze thephosphate group. An example of an agent that cleaves phosphate groups incells are enzymes such as phosphatases in cells. Examples ofphosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—,—O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—,—O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—,—S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—.Additional non-limiting examples are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. Anadditional non-limiting examples is —O—P(O)(OH)—O—.

In some embodiments, a linker comprises an acid cleavable linking groupsare linking groups that are cleaved under acidic conditions. As anon-limiting example, acid cleavable linking groups are cleaved in anacidic environment with a pH of about 6.5 or lower (e.g., about 6.0,5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid. In a cell, specific low pH organelles, such as endosomesand lysosomes can provide a cleaving environment for acid cleavablelinking groups. Examples of acid cleavable linking groups include butare not limited to hydrazones, esters, and esters of amino acids. Acidcleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O).In an additional non-limiting example, when the carbon attached to theoxygen of the ester (the alkoxy group) is an aryl group, substitutedalkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.

In some embodiments, a linker comprises an ester-based linking groups.As a non-limiting example, ester-based cleavable linking groups arecleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

In some embodiments, a linker comprises a peptide-based cleaving group.Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Asa non-limiting example, peptide-based cleavable groups do not includethe amide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynylene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins. Asa non-limiting example, a peptide based cleavage group can be limited tothe peptide bond (i.e., the amide bond) formed between amino acidsyielding peptides and proteins and does not include the entire amidefunctional group. As a non-limiting example, a peptide-based cleavablelinking groups can have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Any linker reported in the art can be used, including, as non-limitingexamples, those described in: U.S. Pat. App. No. 20150265708.

A non-limiting example of a method of conjugating a lipid and anoligonucleotide is presented in Example 1.

A non-limiting example of a linker is a C6 amino linker.

Target Components

In some embodiments, a provided composition further comprises atargeting component (targeting compound or moiety). A target componentcan be either conjugated or not conjugated to a lipid or a biologicallyactive agent. In some embodiments, a target component is conjugated to abiologically active agent. In some embodiments, a biologically activeagent is conjugated to both a lipid and a targeting component. Asdescribed in here, in some embodiments, a biologically active agent is aprovided oligonucleotide. Thus, in some embodiments, a providedoligonucleotide composition further comprises, besides a lipid andoligonucleotides, a target elements. Various targeting components can beused in accordance with the present disclosure, e.g., lipids,antibodies, peptides, carbohydrates, etc.

Target components can be incorporated into provided technologies throughmany types of methods in accordance with the present disclosure. In someembodiments, target components are physically mixed with providedoligonucleotides to form provided compositions. In some embodiments,target components are chemically conjugated with oligonucleotides.

In some embodiments, provided compositions comprise two or more targetcomponents. In some embodiments, provided oligonucleotides comprise twoor more conjugated target components. In some embodiments, the two ormore conjugated target components are the same. In some embodiments, thetwo or more conjugated target components are different. In someembodiments, provided oligonucleotides comprise no more than one targetcomponent. In some embodiments, oligonucleotides of a providedcomposition comprise different types of conjugated target components. Insome embodiments, oligonucleotides of a provided composition comprisethe same type of target components.

Target components can be conjugated to oligonucleotides optionallythrough linkers. Various types of linkers in the art can be utilized inaccordance of the present disclosure. In some embodiments, a linkercomprise a phosphate group, which can, for example, be used forconjugating target components through chemistry similar to thoseemployed in oligonucleotide synthesis. In some embodiments, a linkercomprises an amide, ester, or ether group. In some embodiments, a linkerhas the structure of -L-. Target components can be conjugated througheither the same or different linkers compared to lipids.

Target components, optionally through linkers, can be conjugated tooligonucleotides at various suitable locations. In some embodiments,target components are conjugated through the 5′-OH group. In someembodiments, target components are conjugated through the 3′-OH group.In some embodiments, target components are conjugated through one ormore sugar moieties. In some embodiments, target components areconjugated through one or more bases. In some embodiments, targetcomponents are incorporated through one or more internucleotidiclinkages. In some embodiments, an oligonucleotide may contain multipleconjugated target components which are independently conjugated throughits 5′-OH, 3′-OH, sugar moieties, base moieties and/or internucleotidiclinkages. Target components and lipids can be conjugated either at thesame, neighboring and/or separated locations. In some embodiments, atarget component is conjugated at one end of an oligonucleotide, and alipid is conjugated at the other end.

In some embodiments, the oligonucleotide or oligonucleotides in achirally controlled oligonucleotide composition is or are antisenseoligonucleotide or oligonucleotides. In some embodiments, the sequenceof the oligonucleotide(s) comprises or consists of the sequence of anyoligonucleotide disclosed herein. In some embodiments, providedoligonucleotides comprise base sequence, pattern of backbone linkages,pattern or backbone chiral centers, and/or pattern of chemicalmodifications (e.g., base modifications, sugar modifications, etc.) ofany oligonucleotide disclosed herein. In some embodiments, the sequenceof the oligonucleotide(s) comprises or consists of the sequence of anyoligonucleotide disclosed in Table 8.

In some embodiments, an antisense oligonucleotide is an oligonucleotidewhich participates in RNaseH-mediated cleavage; for example, anantisense oligonucleotide hybridizes in a sequence-specific manner to aportion of a target mRNA, thus targeting the mRNA for cleavage byRNaseH. In some embodiments, an antisense oligonucleotide is able todifferentiate between different alleles of the same gene or target. Insome embodiments, an antisense oligonucleotide is able to differentiatebetween a wild-type and a mutant allele of a target. In someembodiments, an antisense oligonucleotide significantly participates inRNaseH-mediated cleavage of a mutant allele but participates inRNaseH-mediated cleavage of a wild-type allele to a much less degree(e.g., does not significantly participate in RNaseH-mediated cleavage ofthe wild-type allele of the target). In some embodiments, an antisenseoligonucleotide is capable of participating in RNAseH-mediated cleavageof a nucleic acid comprising a mutation. In some embodiments, anantisense oligonucleotide targets a mutant allele. In some embodiments,an antisense oligonucleotide targets a mutant allele of the Huntingtingene.

In some embodiments, an antisense oligonucleotide is able todifferentiate between a wild-type and a mutant allele of a target in theHuntingtin gene.

In some embodiments, the present disclosure pertains to:

A method for inhibiting expression of a mutant Huntingtin gene in amammal comprising preparing a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene) and administeringthe composition to the mammal.

A method of treating a disease that is caused by the over-expression ofa mutant Huntingtin gene in a subject, said method comprising theadministration of a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene).

A method of treating Huntington's Disease, said method comprising theadministration of a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene).

A method for treating a sign and/or symptom of Huntington's Disease in asubject by providing a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene) and administering atherapeutically effective amount of the composition to the subject.

A method of administering an oligonucleotide to a subject in needthereof, comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering the composition to thesubject, wherein the biologically active compound is an oligonucleotide(as a non-limiting example, an antisense oligonucleotide that targets amutant allele of the Huntingtin gene), and wherein the lipid is anylipid disclosed herein.

A method of treating a disease in a subject, the method comprising stepsof providing a composition comprising an oligonucleotide and a lipid,and administering a therapeutically effective amount of the compositionto the subject, wherein the biologically active compound is anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene), and wherein thelipid is any lipid disclosed herein, and wherein the disease is anydisease disclosed herein.

A method for inhibiting expression of a mutant Huntingtin gene in amammal, the method comprising steps of preparing a compositioncomprising a lipid and an oligonucleotide (as a non-limiting example, anantisense oligonucleotide that targets a mutant allele of the Huntingtingene) and administering the composition to the mammal.

A method of administering a biologically active agent to a subject inneed thereof, comprising steps of providing a composition comprising abiologically active agent and a lipid, and administering the compositionto the subject, wherein the biologically active compound is anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene), and wherein thelipid is any lipid disclosed herein.

A method of treating Huntington's Disease in a subject, the methodcomprising steps of providing a composition comprising a biologicallyactive agent and a lipid, and administering a therapeutically effectiveamount of the composition to the subject, wherein the biologicallyactive compound is an oligonucleotide (as a non-limiting example, anantisense oligonucleotide that targets a mutant allele of the Huntingtingene), and wherein the lipid is any lipid disclosed herein.

A method for mediating RNAseH-mediated cleavage of a nucleic acidcomprising a mutant Huntingtin gene in a mammal, the method comprisingsteps of preparing a composition comprising a lipid and an antisenseoligonucleotide and administering the composition to the mammal.

A method of treating a disease that is caused by a mutation in aHuntingtin gene, said method comprising the administration of acomposition comprising a lipid and an antisense oligonucleotide, whereinthe oligonucleotide is capable of participating in RNaseH-mediatedcleavage of a nucleic acid comprising the mutation.

A method for treating a sign and/or symptom of Huntington's Disease in asubject by providing a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene) and administering atherapeutically effective amount of the composition to the subject.

A method of administering an oligonucleotide to a subject in needthereof, comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering the composition to thesubject, wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a nucleic acid comprising a mutation, andwherein the lipid is any lipid disclosed herein.

A method of treating Huntington's Disease in a subject, wherein thedisease or disorder is related to a mutation in a gene, the methodcomprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering a therapeuticallyeffective amount of the composition to the subject, wherein theoligonucleotide is capable of participating in RNaseH-mediated cleavageof a nucleic acid comprising the mutation, and wherein the lipid is anylipid disclosed herein.

A method for mediating RNAseH-mediated cleavage of a nucleic acidcomprising a mutant Huntingtin gene in a mammal, the method comprisingsteps of preparing a composition comprising a lipid and an antisenseoligonucleotide and administering the composition to the mammal.

A method of treating a disease related to a mutation in the Huntingtingene, said method comprising the administration of a compositioncomprising a lipid and an antisense oligonucleotide, wherein theantisense oligonucleotide is capable of participating in RNaseH-mediatedcleavage of a nucleic acid comprising the mutation.

A method of treating a disease that is caused by a mutation in theHuntingtin gene, said method comprising the administration of acomposition comprising a lipid and an oligonucleotide, wherein theoligonucleotide is capable of participating in RNaseH-mediated cleavageof a nucleic acid comprising the mutation.

A method for treating a sign and/or symptom of Huntington's Disease in asubject by providing a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene) and administering atherapeutically effective amount of the composition to the subject.

A method of administering an oligonucleotide to a subject in needthereof, comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering the composition to thesubject, wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a nucleic acid comprising the mutation, andwherein the lipid is any lipid disclosed herein.

A method of treating Huntington's Disease in a subject, wherein theHuntington's Disease is related to a mutation in the Huntingtin gene,the method comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering a therapeuticallyeffective amount of the composition to the subject, wherein theoligonucleotide is capable of participating in RNaseH-mediated cleavageof a nucleic acid comprising the mutation, and wherein the lipid is anylipid disclosed herein.

A method for mediating RNAseH-mediated cleavage of a nucleic acidcomprising a mutant Huntingtin gene in a mammal, the method comprisingsteps of preparing a composition comprising a lipid and an antisenseoligonucleotide and administering the composition to the mammal, whereinthe lipid is any lipid disclosed herein, and wherein the sequence of theantisense oligonucleotide comprises or consists of the sequence of anyantisense oligonucleotide disclosed herein (e.g., in Table 8). In someembodiments, provided oligonucleotides comprise base sequence, patternof backbone linkages, pattern or backbone chiral centers, and/or patternof chemical modifications (e.g., base modifications, sugarmodifications, etc.) of any oligonucleotide disclosed herein (e.g., inTable 8).

A method of treating a disease related to a mutation in the Huntingtingene, said method comprising the administration of a compositioncomprising a lipid and an antisense oligonucleotide, wherein theantisense oligonucleotide is capable of participating in RNaseH-mediatedcleavage of a nucleic acid comprising the mutation, wherein the lipid isany lipid disclosed herein, and wherein the sequence of the antisenseoligonucleotide comprises or consists of the sequence of any antisenseoligonucleotide disclosed herein (e.g., in Table 8).

A method of treating a disease that is caused by a mutation in theHuntingtin gene, said method comprising the administration of acomposition comprising a lipid and an oligonucleotide, wherein theoligonucleotide is capable of participating in RNaseH-mediated cleavageof a nucleic acid comprising the mutation, wherein the lipid is anylipid disclosed herein, and wherein the oligonucleotide comprises orconsists of the sequence of any antisense oligonucleotide disclosedherein (e.g., in Table 8).

A method for treating a sign and/or symptom of Huntington's Disease in asubject by providing a composition comprising a lipid and anoligonucleotide (as a non-limiting example, an antisense oligonucleotidethat targets a mutant allele of the Huntingtin gene) and administering atherapeutically effective amount of the composition to the subject,wherein the lipid is any lipid disclosed herein, and wherein thesequence of the oligonucleotide comprises or consists of the sequence ofany antisense oligonucleotide disclosed herein (e.g., in Table 8).

A method of administering an oligonucleotide to a subject in needthereof, comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering the composition to thesubject, wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a nucleic acid comprising the mutation, andwherein the lipid is any lipid disclosed herein, wherein the lipid isany lipid disclosed herein, and wherein the sequence of theoligonucleotide comprises or consists of the sequence of any antisenseoligonucleotide disclosed herein (e.g., in Table 8).

A method of treating Huntington's Disease in a subject, wherein theHuntington's Disease is related to a mutation in the Huntingtin gene,the method comprising steps of providing a composition comprising anoligonucleotide and a lipid, and administering a therapeuticallyeffective amount of the composition to the subject, wherein theoligonucleotide is capable of participating in RNaseH-mediated cleavageof a nucleic acid comprising the mutation, and wherein the lipid is anylipid disclosed herein, and wherein the oligonucleotide comprises orconsists of the sequence of any antisense oligonucleotide disclosedherein (e.g., in Table 8).

In some embodiments, provided oligonucleotides comprise base sequence,pattern of backbone linkages, pattern or backbone chiral centers, and/orpattern of chemical modifications (e.g., base modifications, sugarmodifications, etc.) of any oligonucleotide disclosed herein.

In some embodiments, an oligonucleotide composition comprises aplurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein one or more oligonucleotides of the plurality are individuallyconjugated to a lipid.

In some embodiments, a chirally controlled oligonucleotide compositioncomprises a plurality of oligonucleotides, which share:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone phosphorus modifications;

wherein:

the composition is chirally controlled in that the plurality ofoligonucleotides share the same stereochemistry at one or more chiralinternucleotidic linkages;

one or more oligonucleotides of the plurality are individuallyconjugated to a lipid; and

one or more oligonucleotides of the plurality are optionally andindividually conjugated to a targeting compound or moiety.

In some embodiments, an antisense oligonucleotide is in a chirallycontrolled oligonucleotide composition. In some embodiments, anoligonucleotide is in a chirally controlled oligonucleotide composition.

Various oligonucleotides are listed in Table 8. Many of these arecapable of participating in RNaseH-mediated cleavage of the humanHuntingtin gene, as shown in data presented in U.S. Pat. Application No.62/195,779, filed Jul. 22, 2015, and U.S. Pat. Application No.62/331,960, filed May 4, 2016, which are incorporated by reference inits entirety; and in data shown here.

Various oligonucleotides particularly capable of participating inRNaseH-mediated cleavage of human Huntingtin gene target or a mutantvariant thereof include: WV-1092, WVE120101, WV-2603 or WV-2595, or anyother nucleic acid disclosed herein (including, but not limited to,those listed in Table 8).

Any oligonucleotide or chirally controlled oligonucleotide compositioncan be used in combination with any method or composition (e.g., anypharmaceutical composition, modification, and/or method of use and/ormanufacture) disclosed herein.

In some embodiments, the present disclosure provides the followingembodiments:

1. A chirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

2. A chirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.2a. A chirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type, wherein the oligonucleotides target amutant Huntingtin gene, and the length is from about 10 to about 50nucleotides, wherein the backbone linkages comprise at least onephosphorothioate, and wherein the pattern of backbone chiral centerscomprises at least one chiral center in a Rp conformation and at leastone chiral center in a Sp conformation.3. A chirally controlled oligonucleotide composition comprisingoligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that atleast about 10% of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

4. A composition of any one of the preceding embodiments, wherein theoligonucleotides comprise one or more wing regions and a common coreregion, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more chiral internucleotidic linkages.

5. An oligonucleotide composition comprising a predetermined level ofoligonucleotides which comprise one or more wing regions and a commoncore region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

6. An oligonucleotide composition comprising a predetermined level ofoligonucleotides which comprise one or more wing regions and a commoncore region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the core region has:

1) a common base sequence;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers; and

4) a common pattern of backbone phosphorus modifications.

6a. A composition of any one of the preceding embodiments, whereinoligonucleotides of the oligonucleotide type comprises at least one wingregion and a core region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages;and

wherein at least one nucleotide in a wing region differs from at leastone nucleotide of the core region, wherein the difference is in one ormore of:

1) backbone linkage;

2) pattern of backbone chiral centers;

3) sugar modification.

7. A composition of any one of the preceding embodiments, wherein theoligonucleotides are defined by having a common pattern of backbonephosphorus modifications.8. A composition of any one of the preceding embodiments, wherein thecomposition contains a predetermined level of oligonucleotides of anindividual oligonucleotide type, wherein an oligonucleotide type isdefined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

9. A composition of any one of the preceding embodiments, whereinoligonucleotides having a common base sequence is of the sameoligonucleotide type characterized by base sequence, pattern of backbonelinkages, pattern of backbone chiral centers, and pattern of backbonephosphorus modifications.10. A composition of any one of the preceding embodiments, wherein thecomposition contains predetermined levels of oligonucleotides of two ormore individual oligonucleotide types, wherein an oligonucleotide typeis defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

11. An oligonucleotide composition that is chirally controlled in thatthe composition contains a predetermined level of oligonucleotides of anindividual oligonucleotide type, wherein an oligonucleotide type isdefined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

12. A composition of any one of the preceding embodiments, wherein thecomposition comprises two or more individual oligonucleotide types.13. A composition of any one of the preceding embodiments, wherein anoligonucleotide type is defined by base identity, pattern of basemodification, pattern of sugar modification, pattern of backbonelinkages, pattern of backbone chiral centers, and pattern of backbonephosphorus modifications.14. A composition of any one of the preceding embodiments, whereinoligonucleotides having a common sequence have identical structure.15. A composition of any one of the preceding embodiments, whereinoligonucleotides of the same oligonucleotide type have identicalstructure.16. A composition of any one of the preceding embodiments, wherein theoligonucleotides have one wing.17. A composition of any one of the preceding embodiments, wherein theoligonucleotides are hemimers having the structure of wing-core.18. A composition of any one of the preceding embodiments, wherein theoligonucleotides are hemimers having the structure of core-wing.19. A composition of any one of embodiments 1-15, wherein theoligonucleotides have two wings.20. A composition of any one of embodiments 1-15, wherein theoligonucleotides are gapmers having the structure of wing-core-wing.21. A composition of any one of the preceding embodiments, wherein awing comprises a chiral internucleotidic linkage.22. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a chiral internucleotidic linkage.23. A composition of any one of embodiments 1-17 and 19-22, wherein awing to the 5′-end of the core comprises a chiral internucleotidiclinkage at the 5′-end of the wing.24. A composition of any one of embodiments 1-16 and 18-22, wherein awing to the 3′-end of the core comprises a chiral internucleotidiclinkage at the 3′-end of the wing.25. A composition of any one the preceding embodiments, wherein a winghas only one chiral internucleotidic linkage, and each of the otherinternucleotidic linkages of the wing is a natural phosphate linkage

26. A composition of any one of the preceding embodiments, wherein thechiral internucleotidic linkage has the structure of formula I.27. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage has the structure of formula I, andwherein X is S, and Y and Z are O.28. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage is a phosphorothioate linkage.29. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage is Sp.30. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is Sp.31. A composition of any one of embodiments 1-29, wherein a chiralinternucleotidic linkage is Rp.32. A composition of any one of embodiments 1-28, wherein each chiralinternucleotidic linkage is Rp.33. A composition of any one of embodiments 1-31, wherein a wingcomprises an Sp phosphorothioate linkage.34. A composition of any one of embodiments 1-31, wherein each wingindependently comprises an Sp phosphorothioate linkage.35. A composition of any one of embodiments 1-17, 19-31, and 33-34,wherein a wing is to the 5′-end of the core, and the wing has an Spphosphorothioate linkage.36. A composition of any one of embodiments 1-17, 19-31, and 33-35,wherein a wing is to the 5′-end of the core, and the wing has an Spphosphorothioate linkage at the 5′-end of the wing.37. A composition of any one of embodiments 1-17, 19-31, and 33-36,wherein a wing is to the 5′-end of the core, the wing has an Spphosphorothioate linkage at the 5′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

38. A composition of any one of embodiments 1-16, 18-31 and 33-34,wherein a wing is to the 3′-end of the core, and the wing has an Spphosphorothioate linkage at the 3′-end of the wing.39. A composition of any one of embodiments 1-16, 18-31, 33-34 and 38,wherein a wing is to the 3′-end of the core, and the wing has an Spphosphorothioate linkage at the 3′-end of the wing.40. A composition of any one of embodiments 1-16, 18-31, 33-34 and38-39, wherein one wing is to the 3′-end of the common core, the winghas an Sp phosphorothioate linkage at the 3′-end of the wing, and eachof the other internucleotidic linkages of the wing is a naturalphosphate linkage

41. A composition of any one of embodiments 1-29 and 31-40, wherein awing comprises an Rp phosphorothioate linkage.42. A composition of any one of embodiments 1-29 and 31-40, wherein eachwing independently comprises an Rp phosphorothioate linkage.43. A composition of any one of embodiments 1-17, 19-29 and 31-42,wherein a wing is to the 5′-end of the core, and the wing has an Rpphosphorothioate linkage.44. A composition of any one of embodiments 1-17, 19-29 and 31-43,wherein a wing is to the 5′-end of the core, and the wing has an Rpphosphorothioate linkage at the 5′-end of the wing.45. A composition of any one of embodiments 1-17, 19-29 and 31-44,wherein a wing is to the 5′-end of the core, the wing has an Rpphosphorothioate linkage at the 5′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

46. A composition of any one of embodiments 1-16, 18-29 and 31-42,wherein a wing is to the 3′-end of the core, and the wing has an Rpphosphorothioate.47. A composition of any one of embodiments 1-16, 18-29 and 31-42,wherein a wing is to the 3′-end of the core, and the wing has an Rpphosphorothioate linkage at the 3′-end of the wing.48. A composition of any one of embodiments 1-16, 18-29 and 31-42,wherein one wing is to the 3′-end of the common core, the wing has an Rpphosphorothioate linkage at the 3′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

49. A composition of any one of embodiments 1-28, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is achiral internucleotidic linkage.50. A composition of any one of embodiments 1-28, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is an Spchiral internucleotidic linkage.51. A composition of any one of embodiments 1-28, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is an Rpchiral internucleotidic linkage.52. A composition of any one of embodiments 1-28 and 49-51, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis a chiral internucleotidic linkage.53. A composition of any one of embodiments 1-28 and 49-51, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis an Sp chiral internucleotidic linkage.54. A composition of any one of embodiments 1-28 and 49-51, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis an Rp chiral internucleotidic linkage.55. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a natural phosphate linkage

56. A composition of any one of the preceding embodiments, wherein eachwing independently comprises two or more natural phosphate linkages

57. A composition of any one of the preceding embodiments, wherein eachwing independently comprises two or more natural phosphate linkages, andall natural phosphate linkages are consecutive.58. A composition of any one of the preceding embodiments, wherein awing has a length of three or more bases.59. A composition of any one of the preceding embodiments, wherein onewing has a length of four or more bases.60. A composition of any one of the preceding embodiments, wherein onewing has a length of five or more bases.61. A composition of any one of the preceding embodiments, wherein onewing has a length of six or more bases.62. A composition of any one of the preceding embodiments, wherein onewing has a length of seven or more bases.63. A composition of any one of the preceding embodiments, wherein onewing has a length of eight or more bases.64. A composition of any one of the preceding embodiments, wherein onewing has a length of nine or more bases.65. A composition of any one of the preceding embodiments, wherein onewing has a length of ten or more bases.66. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of three or more bases.67. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of four or more bases.68. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of five or more bases.69. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of six or more bases.70. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of seven or more bases.71. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of eight or more bases.72. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of nine or more bases.73. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of ten or more bases.74. A composition of any one of embodiments 1-57, wherein a wing has alength of two bases.75. A composition of any one of embodiments 1-57, wherein a wing has alength of three bases.76. A composition of any one of embodiments 1-57, wherein a wing has alength of four bases.77. A composition of any one of embodiments 1-57, wherein a wing has alength of five bases.78. A composition of any one of embodiments 1-57, wherein a wing has alength of six bases.79. A composition of any one of embodiments 1-57, wherein a wing has alength of seven bases.80. A composition of any one of embodiments 1-57, wherein a wing has alength of eight bases.81. A composition of any one of embodiments 1-57, wherein a wing has alength of nine bases.82. A composition of any one of embodiments 1-57, wherein a wing has alength of ten bases.83. A composition of any one of embodiments 1-57, wherein a wing has alength of 11 bases.84. A composition of any one of embodiments 1-57, wherein a wing has alength of 12 bases.85. A composition of any one of embodiments 1-57, wherein a wing has alength of 13 bases.86. A composition of any one of embodiments 1-57, wherein a wing has alength of 14 bases.87. A composition of any one of embodiments 1-57, wherein a wing has alength of 15 bases.88. A composition of any one of embodiments 1-11 and 15-76, wherein eachwing has the same length.89. A composition of any one of the preceding embodiments, wherein awing is defined by sugar modifications relative to a core.90. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a modified sugar moiety.91. A composition of any one of the preceding embodiments, wherein eachwing sugar moiety is independently a modified sugar moiety.92. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a high-affinity sugar modification.93. A composition of any one of the preceding embodiments, wherein amodified sugar moiety has a 2′-modification.94. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification.95. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification having a-L- or —O-L- bridge connecting two ring carbon atoms.96. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification having a4′-CH(CH₃)—O-2′ bridge.97. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OR¹.98. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OR¹, wherein R¹ is optionally substituted C₁₋₆ alkyl.99. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-MOE.100. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OMe.101. A composition of any one of embodiments 1-96, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isS-cEt.102. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isFANA.103. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isFRNA.104. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety has a 5′-modification.105. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is R-5′-Me-DNA.106. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is S-5′-Me-DNA.107. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is FHNA.108. A composition of any one of the preceding embodiments, wherein eachwing sugar moiety is modified.109. A composition of any one of the preceding embodiments, wherein allmodified wing sugar moieties within a wing have the same modification.110. A composition of any one of the preceding embodiments, wherein allmodified wing sugar moieties have the same modification.111. A composition of any one of embodiments 1-108, wherein at least onemodified wing sugar moiety is different than another modified wing sugarmoiety.112. A composition of any one of the preceding embodiments, wherein awing comprises a modified base.113. A composition of any one of the preceding embodiments, wherein awing comprises a 2S-dT.114. A composition of any one of the preceding embodiments, wherein thecore region has a length of five or more bases.115. A composition of any one of the preceding embodiments, wherein thecore region has a length of six or more bases.116. A composition of any one of the preceding embodiments, wherein thecore region has a length of seven or more bases.117. A composition of any one of the preceding embodiments, wherein thecore region has a length of eight or more bases.118. A composition of any one of the preceding embodiments, wherein thecore region has a length of nine or more bases.119. A composition of any one of the preceding embodiments, wherein thecore region has a length of ten or more bases.120. A composition of any one of the preceding embodiments, wherein thecore region has a length of 11 or more bases.121. A composition of any one of the preceding embodiments, wherein thecore region has a length of 12 or more bases.122. A composition of any one of the preceding embodiments, wherein thecore region has a length of 13 or more bases.123. A composition of any one of the preceding embodiments, wherein thecore region has a length of 14 or more bases.124. A composition of any one of the preceding embodiments, wherein thecore region has a length of 15 or more bases.125. A composition of any one of 1-113, wherein the core region has alength of five bases.126. A composition of any one of 1-113, wherein the core region has alength of six bases.127. A composition of any one of 1-113, wherein the core region has alength of seven bases.128. A composition of any one of 1-113, wherein the core region has alength of eight bases.129. A composition of any one of 1-113, wherein the core region has alength of nine bases.130. A composition of any one of 1-113, wherein the core region has alength of ten bases.131. A composition of any one of 1-113, wherein the core region has alength of 11 bases.132. A composition of any one of 1-113, wherein the core region has alength of 12 bases.133. A composition of any one of 1-113, wherein the core region has alength of 13 bases.134. A composition of any one of 1-113, wherein the core region has alength of 14 bases.135. A composition of any one of 1-113, wherein the core region has alength of 15 bases.136. A composition of any one of the preceding embodiments, wherein thecore region does not have any 2′-modification.137. A composition of any one of the preceding embodiments, wherein eachcore sugar moiety is not modified.138. A composition of any one of the preceding embodiments, wherein eachsugar moiety of the core region is the natural DNA sugar moiety.139. A composition of any one of the preceding embodiments, wherein thecore region comprises a chiral internucleotidic linkage.140. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage.141. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I.142. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I, and wherein X is S, and Y andZ are O.143. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I, and wherein one -L-R¹ is not—H.144. A composition of any one of embodiments 1-142, wherein eachinternucleotidic linkage of the core region is a phosphorothioatelinkage.145. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)_(m)(Rp)_(n), wherein m is 1-50, and n is 1-10.146. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)_(m)(Rp)_(n), wherein m is 1-50, n is 1-10, and m>n.147. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)_(m)(Rp)_(n), wherein m is 2, 3, 4, 5, 6, 7 or 8, and n is 1.148. A composition of any one of embodiments 1-144, wherein the coreregion has a pattern of backbone chiral centers comprising(Rp)_(n)(Sp)_(m), wherein m is 1-50 and n is 1-10.149. A composition of any one of embodiments 1-144 and 148, wherein thecore region has a pattern of backbone chiral centers comprisingRp(Sp)_(m), wherein m is 2, 3, 4, 5, 6, 7 or 8.150. A composition of any one of embodiments 1-144 and 148-149, whereinthe core region has a pattern of backbone chiral centers comprisingRp(Sp)₂.151. A composition of any one of embodiments 1-144, wherein the coreregion has a pattern of backbone chiral centers comprising(Np)_(t)(Rp)_(n)(Sp)_(m), wherein t is 1-10, n is 1-10, m is 1-50, andeach Np is independent Rp or Sp.152. A composition of any one of embodiments 1-144 and 151, wherein thecore region has a pattern of backbone chiral centers comprising(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein t is 1-10, n is 1-10, m is 1-50.153. A composition of any one of embodiments 1-144 and 151-152, whereinn is 1.154. A composition of any one of embodiments 1-144 and 151-153, whereint is 2, 3, 4, 5, 6, 7 or 8.155. A composition of any one of embodiments 1-144 and 151-154, whereinm is 2, 3, 4, 5, 6, 7 or 8.156. A composition of any one of embodiments 1-144 and 151-155, whereinat least one of t and m is greater than 5.157. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral centers comprisingSpSpRpSpSp.158. A composition of any one of the preceding embodiments, wherein 50%or more of the chiral internucleotidic linkages in the core region haveSp configuration.159. A composition of any one of the preceding embodiments, wherein 60%or more of the chiral internucleotidic linkages in the core region haveSp configuration.160. A composition of any one of the preceding embodiments, wherein 70%or more of the chiral internucleotidic linkages in the core region haveSp configuration.161. A composition of any one of the preceding embodiments, wherein 80%or more of the chiral internucleotidic linkages in the core region haveSp configuration.162. A composition of any one of the preceding embodiments, wherein 90%or more of the chiral internucleotidic linkages in the core region haveSp configuration.163. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage in the core region is chiral, the core regionhas only one Rp, and each of the other internucleotidic linkages in thecore region is Sp.164. A composition of any one of the preceding embodiments, wherein eachbase moiety in the core is not modified.165. A composition of any one of embodiments 1-163, wherein the coreregion comprises a modified base.166. A composition of any one of embodiments 1-163, wherein the coreregion comprises a modified base, wherein a modified base is substitutedA, T, C or G.167. A composition of any one of embodiments 1-164, wherein each basemoiety in the core region is independently selected from A, T, C and G.168. A composition of any one of embodiments 1-163, wherein the coreregion is a DNA sequence whose phosphate linkages are independentlyreplaced with phosphorothioate linkages.169. A composition of any one of the preceding embodiments, wherein theoligonucleotides are single stranded.170. A composition of any one of the preceding embodiments, wherein theoligonucleotides are antisense oligonucleotide, antagomir, microRNA,pre-microRNs, antimir, supermir, ribozyme, Ul adaptor, RNA activator,RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide,aptamer or adjuvant.171. A composition of any one of the preceding embodiments, wherein theoligonucleotides are antisense oligonucleotides.172. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 10 bases.173. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 11 bases.174. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 12 bases.175. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 13 bases.176. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 14 bases.177. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 15 bases.178. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 16 bases.179. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 17 bases.180. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 18 bases.181. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 19 bases.182. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 20 bases.183. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 21 bases.184. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 22 bases.185. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 23 bases.186. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 24 bases.187. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 25 bases.188. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 200 bases.189. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 150 bases.190. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 100 bases.191. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 50 bases.192. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 40 bases.193. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 30 bases.194. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 10 bases.195. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 11 bases.196. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 12 bases.197. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 13 bases.198. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 14 bases.199. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 15 bases.200. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 16 bases.201. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 17 bases.202. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 18 bases.203. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 19 bases.204. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 20 bases.205. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 21 bases.206. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 22 bases.207. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 23 bases.208. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 24 bases.209. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 25 bases.210. A composition of any one of the preceding embodiments, wherein theoligonucleotide type is not (Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 1505) or(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC(5R-(SSR)3-5R) (SEQ ID NO: 1506), wherein in the underlined nucleotideare 2′-MOE modified.211. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from: (Sp, Sp, Rp,Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 1505) or(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC(5R-(SSR)3-5R) (SEQ ID NO: 1506), wherein in the underlined nucleotideare 2′-MOE modified.212. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from:

ONT-106 (SEQ ID NO: 1507) PCSK9 sense (Rp)-uucuAGAccuGuuuuGcuudTsdTONT-107 (SEQ ID NO: 1508) PCSK9 sense (Sp)-uucuAGAccuGuuuuGcuudTsdTONT-108 (SEQ ID NO: 1509) PCSK9 antisense (Rp)-AAGcAAAAcAGGUCuAGAAdTsdTONT-109 (SEQ ID NO: 1510) PCSK9 antisense (Sp)-AAGcAAAAcAGGUCuAGAAdTsdTONT-110 (SEQ ID NO: 1511) PCSK9 antisense (Rp,Rp)-asAGcAAAAcAGGUCuAGAAdTsdT ONT-111 (SEQ ID NO: 1512) PCSK9 antisense(Sp, Rp)-asGcAAAAcAGGUCuAGAAdTsdT ONT-112 (SEQ ID NO: 1513) PCSK9antisense (Sp, Sp)-asGcAAAAcAGGUCuAGAAdTsdT ONT-113 (SEQ ID NO: 1514)PCSK9 antisense (Rp, Sp)-asGcAAAAcAGGUCuAGAAdTsdTwherein lower case letters represent 2′OMe RNA residues; capital lettersrepresent 2′OH RNA residues; and bolded and “s” indicates aphosphorothioate moiety; and

PCSK9 (1) (SEQ ID NO: 1515) (All(Sp))-ususcsusAsGsAscscsusGsususususGscsususd TsdT PCSK9 (2) (SEQ ID NO:1516) (All (Rp))-ususcsusAsGsAscscsusGsususususGscsususd TsdT PCSK9 (3)(SEQ ID NO: 1517) (All (Sp))-usucuAsGsAsccuGsuuuuGscuusdTsdT PCSK9 (4)(SEQ ID NO: 1518) (All (Rp))-usucuAsGsAsccuGsuuuuGscuusdTsdT PCSK9 (5)(SEQ ID NO: 1519) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp)-ususcsusAsGsAscscs usGsususususGscsususdTsdTPCSK9 (6) (SEQ ID NO: 1520) (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-ususcsusAsGsAscscsusGsususususGscsususdTsdTwherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d=2′-deoxy residues; and “s” indicates aphosphorothioate moiety; and

PCSK9 (7) (SEQ ID NO: 1521) (All(Rp))-AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGsAsAsd TsdT PCSK9 (8) (SEQ ID NO:1522) (All (Sp))-AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGsAsAsd TsdT PCSK9 (9)(SEQ ID NO: 1523) (All (Rp))-AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdTsdT PCSK9(10) (SEQ ID NO: 1524) (All (Sp))-AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdTsdTPCSK9 (11) (SEQ ID NO: 1525) (All (Rp))-AAsGscsAsAsAsAscAGGUCuAGAAdTsdTPCSK9 (12) (SEQ ID NO: 1526) (All (Sp))-AAsGscsAsAsAsAscAGGUCuAGAAdTsdTPCSK9 (13) (SEQ ID NO: 1527) (All(Rp))-AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsAsdTsd T PCSK9 (14) (SEQ ID NO:1528) (All (Sp))-AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsAsdTsd T PCSK9 (15)(SEQ ID NO: 1529) (All (Rp))-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9(16) (SEQ ID NO: 1530) (All (Sp))-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdTPCSK9 (17) (SEQ ID NO: 1531) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT PCSK9 (18) (SEQ IDNO: 1532) (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdTwherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d=2′-deoxy residues; “s” indicates aphosphorothioate moiety; and

PCSK9 (19) (SEQ ID NO: 1533) (All(Rp))-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsg sCfsusUfsdTsdT PCSK9 (20)(SEQ ID NO: 1534) (All (Sp))-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT PCSK9 (21) (SEQ ID NO: 1535) (All(Rp))-UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfsuUfs dTsdT PCSK9 (22) (SEQ IDNO: 1536) (All (Sp))-UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfsuUfs dTsdT PCSK9(23) (SEQ ID NO: 1537) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT PCSK9 (24) (SEQ ID NO: 1538) (Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-UfsusCfsusAfsgsAfs csCfsusGfsusUfsusUfsgsCfsusUfsdTsdTwherein lower case letters represent 2′-OMe RNA residues; capitalletters represent 2′-F RNA residues; d=2′-deoxy residues; and “s”indicates a phosphorothioate moiety; and

PCSK9 (25) (SEQ ID NO: 1539) (All(Rp))-asAfsgsCfsasAfsasAfscsAfsgsGfsusCfsusAf sgsAfsasdTsdT PCSK9 (26)(SEQ ID NO: 1540) (All (Sp))-asAfsgsCfsasAfsasAfscsAfsgsGfsusCfsusAfsgsAfsasdTsdT PCSK9 (27) (SEQ ID NO: 1541) (All(Rp))-asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsgsAfsa sdTsdT PCSK9 (28) (SEQ IDNO: 1542) (All (Sp))-asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsgsAfsa sdTsdTPCSK9 (29) (SEQ ID NO: 1543) (All(Rp))-asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsgAfsad TsdT PCSK9 (30) (SEQ IDNO: 1544) (All (Sp))-asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsgAfsad TsdT PCSK9(31) (SEQ ID NO: 1544) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp)-asAfgCfaAfasAfscAfsgsGfsusCfsusAfsgsAfsasd TsdT PCSK9 (32) (SEQID NO: 1546) (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-asAfgCfaAfasAfscAfsgsGfsusCfsusAfsgsAfsasd TsdT213. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from:d[A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C] (SEQ ID NO: 1555),d[C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C_(S)C_(S)C] (SEQ ID NO: 1556),d[C_(S)C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C] (SEQ ID NO: 1557) andd[C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C] (SEQ ID NO: 1558),wherein R is Rp phosphorothioate linkage, and S is Sp phosphorothioatelinkage.214. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from:GGA_(R)T_(S)G_(R)T_(S)T_(R) ^(m)C_(S)TCGA (SEQ ID NO: 1547),GGA_(R)T_(R)G_(S)T_(S)T_(R) ^(m)C_(R)TCGA (SEQ ID NO: 1548),GGA_(S)T_(S)G_(R)T_(R)T_(S) ^(m)C_(S)TCGA (SEQ ID NO: 1549), wherein Ris Rp phosphorothioate linkage, S is Sp phosphorothioate linkage, allother linkages are PO, and each ^(m)C is a 5-methylcytosine modifiednucleoside.215. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from: T_(k)T_(k)^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 1550),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methylcytosine modified nucleoside, and allinternucleoside linkages are phosphorothioates (PS) with stereochemistrypatterns selected from RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR,RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS,SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.215a. A composition of any one of the preceding embodiments, wherein thecommon pattern of backbone chiral centers comprises SSR, RSS, SSRSS,SSRSSR, RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS,RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR,RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR,RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS,RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, orSSSSRRRRSR.215b. A composition of any one of the preceding embodiments, wherein thecommon pattern of backbone chiral centers comprises SSRSS, SSRSSR,RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR,RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS,SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR,RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR,RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, or SSSSRRRRSR.215c. A composition of any one of the preceding embodiments, wherein thecommon pattern of backbone chiral centers comprises RSSSRSRRRS,RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR,SRSSSRSSSS, SSRRSSRSRS, SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS,SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR,RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR,RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, or SSSSRRRRSR.216. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from: T_(k)T_(k)^(m)C_(k) AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 1551),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methylcytosine modified nucleoside and allinternucleoside linkages in the underlined core are phosphorothioates(PS) with stereochemistry patterns selected from: RSSSRSRRRS,RSSSSSSSSS, SRRSRSSSSR, SRSRSSR SRSSRSS, RRRSRSSRSR, RRSSSRSRSR,SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR,RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR,RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS,RSRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.217. A composition of embodiment 215 or 216, wherein eachphosphorothioate moiety of each nucleotide comprising (S)-cEtmodification is stereorandom.218. A composition of any one of the preceding embodiments, wherein thebase sequence is or comprises a sequence that is complementary to atarget sequence, wherein when contacted with a nucleic acid polymercomprising the target sequence, the composition provides an alteredcleavage pattern than a reference cleavage pattern from a referenceoligonucleotide composition.219. A composition of any one of the preceding embodiments, wherein thenucleic acid polymer is RNA, and a reference oligonucleotide compositionis a substantially racemic preparation of oligonucleotides that sharethe common sequence and length.220. A composition of any one of the preceding embodiments, wherein thenucleic acid polymer is RNA, and a reference oligonucleotide compositionis a chirally uncontrolled oligonucleotide composition ofoligonucleotides that share the common sequence and length.221. A composition of any one of the preceding embodiments, wherein thealtered cleavage pattern has fewer cleavage sites than the referencecleavage pattern.222. A composition of any one of the preceding embodiments, wherein thealtered cleavage pattern has only one cleavage site within the targetsequence, and the reference cleavage pattern has two or more cleavagesites within the target sequence.223. A composition of any one of the preceding embodiments, wherein thebase sequence for the oligonucleotides is or comprises a sequence thatis complementary to a characteristic sequence element that defines aparticular allele of a target gene relative to other alleles of the sametarget gene that exist in a population, the composition beingcharacterized in that, when it is contacted with a system expressingtranscripts of both the target allele and another allele of the samegene, transcripts of the particular allele are suppressed at a level atleast 2 fold greater than a level of suppression observed for anotherallele of the same gene.224. A composition of any one of the preceding embodiments, wherein thebase sequence for the oligonucleotides is or comprises a sequence thatis complementary to a characteristic sequence element that defines aparticular allele of a target gene relative to other alleles of the sametarget gene that exist in a population, the composition beingcharacterized in that, when it is contacted with a system expressingtranscripts of the target gene, it shows suppression of expression oftranscripts of the particular allele at a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

225. A composition of any one of the preceding embodiments, wherein thebase sequence comprises a sequence that is complementary to acharacteristic sequence element of a target, wherein a characteristicsequence element defines that target sequence relative to a similarsequence.226. A composition of any one of the preceding embodiments, wherein thebase sequence of a core region comprises a sequence that iscomplementary to a characteristic sequence element of a target, whereina characteristic sequence element defines that target sequence relativeto a similar sequence.227. A composition of any one of the preceding embodiments, wherein atarget sequence is a sequence comprising a mutation, and a similarsequence is the wild-type sequence.228. A composition of any one of the preceding embodiments, wherein acharacteristic sequence element defines a particular allele of a targetsequence relative to other alleles of the same target sequence.229. A composition of any one of the preceding embodiments, wherein acharacteristic sequence element defines a particular allele of a targetgene relative to other alleles of the same target gene.230. A composition of any one of the preceding embodiments, wherein thesequence is 100% complementary to a characteristic sequence element.231. A composition of any one of the preceding embodiments, whereinposition 11, 12, or 13 of the oligonucleotides as counted from the5′-terminus of the oligonucleotides aligns with a characteristicsequence element.232. A composition of any one of embodiments 1-230, wherein position 11of the oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a characteristic sequence element.233. A composition of any one of embodiments 1-230, wherein position 12of the oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a characteristic sequence element.234. A composition of any one of embodiments 1-230, wherein position 13of the oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a characteristic sequence element.235. A composition of any one of embodiments 1-230, wherein position 8,9 or 10 of the oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a characteristic sequence element.236. A composition of any one of embodiments 1-230, wherein position 8of the oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a characteristic sequence element.237. A composition of any one of embodiments 1-230, wherein position 9of the oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a characteristic sequence element.238. A composition of any one of embodiments 1-230, wherein position 10of the oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a characteristic sequence element.239. A composition of any one of embodiments 1-230, wherein position 6,7 or 8 of the core region as counted from the 5′-terminus of the coreregion aligns with a characteristic sequence element.240. A composition of any one of embodiments 1-230, wherein position 6of the core region as counted from the 5′-terminus of the core regionaligns with a characteristic sequence element.241. A composition of any one of embodiments 1-230, wherein position 7of the core region as counted from the 5′-terminus of the core regionaligns with a characteristic sequence element.242. A composition of any one of embodiments 1-230, wherein position 8of the core region as counted from the 5′-terminus of the core regionaligns with a characteristic sequence element.243. A composition of any one of embodiments 1-230, wherein position 3,4 or 5 of the core region as counted from the 3′-terminus of the coreregion aligns with a characteristic sequence element.244. A composition of any one of embodiments 1-230, wherein position 3of the core region as counted from the 3′-terminus of the core regionaligns with a characteristic sequence element.245. A composition of any one of embodiments 1-230, wherein position 4of the core region as counted from the 3′-terminus of the core regionaligns with a characteristic sequence element.246. A composition of any one of embodiments 1-230, wherein position 5of the core region as counted from the 3′-terminus of the core regionaligns with a characteristic sequence element.247. A composition of any one of the preceding embodiments, wherein acommon base sequence or a base sequence of an oligonucleotide type is asequence whose DNA cleavage pattern has a cleavage site within or in thevicinity of a characteristic sequence element of a target nucleic acidsequence.248. A composition of any one of the preceding embodiments, wherein theDNA cleavage pattern is the cleavage pattern of an oligonucleotidecomposition of DNA oligonucleotides having the sequence, wherein eacholigonucleotide in the composition has the same structure.249. A composition of any one of the preceding embodiments, wherein acommon base sequence or a base sequence of an oligonucleotide type is asequence whose stereorandom cleavage pattern has a cleavage site withinor in the vicinity of a characteristic sequence element of a targetnucleic acid sequence.250. A composition of any one of the preceding embodiments, wherein thestereorandom cleavage pattern is the cleavage pattern of a stereorandomcomposition of oligonucleotides having the sequence, wherein eachinternucleotidic linkage is phosphorothioate.251. A composition of any one of the preceding embodiments, wherein thecleavage site with or in the vicinity of a characteristic sequenceelement of a target nucleic acid sequence is in a core region.252. A composition of any one of the preceding embodiments, wherein thecleavage site is in the vicinity of a characteristic sequence element ofa target nucleic acid sequence.253. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 0, 1, 2, 3, 4, or 5internucleotidic linkages away from the characteristic sequence element.254. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 0 internucleotidiclinkages away from the characteristic sequence element.255. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 1 internucleotidiclinkage away from the characteristic sequence element.256. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 2 internucleotidiclinkages away from the characteristic sequence element.257. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 3 internucleotidiclinkages away from the characteristic sequence element.258. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 4 internucleotidiclinkages away from the characteristic sequence element.259. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site 5 internucleotidiclinkages away from the characteristic sequence element.260. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site is 5′ to the cleavagesite.261. A composition of any one of the preceding embodiments, wherein acleavage site in the vicinity is a cleavage site is 3′ to the cleavagesite.261a. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a major cleavage site.262. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site.263. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 40% oftotal cleavage occurs at the site.264. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 50% oftotal cleavage occurs at the site.265. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 60% oftotal cleavage occurs at the site.266. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 70% oftotal cleavage occurs at the site.267. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 80% oftotal cleavage occurs at the site.268. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 90% oftotal cleavage occurs at the site.269. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 95% oftotal cleavage occurs at the site.270. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is a relative major cleavage site, wherein greater than 100% oftotal cleavage occurs at the site.271. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 5% oftotal target is cleaved at the site.272. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 10% oftotal target is cleaved at the site.273. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 15% oftotal target is cleaved at the site.274. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 20% oftotal target is cleaved at the site.275. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 25% oftotal target is cleaved at the site.276. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 30% oftotal target is cleaved at the site.277. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 35% oftotal target is cleaved at the site.278. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 40% oftotal target is cleaved at the site.279. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 45% oftotal target is cleaved at the site.280. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 50% oftotal target is cleaved at the site.281. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 60% oftotal target is cleaved at the site.282. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 70% oftotal target is cleaved at the site.283. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 80% oftotal target is cleaved at the site.284. A composition of any one of the preceding embodiments, wherein acleavage site within or in the vicinity of a characteristic sequenceelement is an absolute major cleavage site, wherein greater than 90% oftotal target is cleaved at the site.285. A composition of any one the preceding embodiments, wherein arelative or absolute major cleavage site is determined by RNase H assay.286. A composition of any one of the preceding embodiments, wherein thecharacteristic sequence element comprises a single nucleotidepolymorphism (SNP) or a mutation.287. A composition of any one of the preceding embodiments, wherein thecharacteristic sequence element comprises a single nucleotidepolymorphism.288. A composition of any one of the preceding embodiments, wherein thecharacteristic sequence element is a single nucleotide polymorphism.289. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is a single nucleotide polymorphismassociated with Huntington's disease.290. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is a single nucleotide polymorphism foundin the Huntingtin gene.291. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is selected from rs362307, rs7685686,rs362268, rs2530595, rs362331, or rs362306.291a. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is selected from rs362307, rs7685686,rs362268, or rs362306.292. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs362307.293. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs7685686.294. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs362268.295. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs362306.295a. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs2530595.295b. A composition of any one of the preceding embodiments, wherein thesingle nucleotide polymorphism is rs362331.296. A composition of any one of embodiments 1-290, wherein the singlenucleotide polymorphism is in an exon.297. A composition of any one of embodiments 1-290, wherein the singlenucleotide polymorphism is in an intron.298. A composition of any one of embodiments 1-290, wherein thecomposition is selected from Tables N1, N2, N3, N4 and 8.298a. A composition of any one of embodiments 1-290, wherein thecomposition is selected from Tables N1, N2, N3 and N4.299. A composition of any one of embodiments 1-290, wherein thecomposition is selected from Tables N1A, N2A, N3A, N4A and 8; andWV-1092, WVE120101, WV-2603 and WV-2595.299a. A composition of any one of embodiments 1-290, wherein thecomposition is selected from Tables N1A, N2A, N3A and N4A.300. A composition of any one of embodiments 1-290, wherein thecomposition is WV-1092.300a. A composition of any one of embodiments 1-290, wherein thecomposition is WVE120101.300b. A composition of any one of embodiments 1-290, wherein thecomposition is WV-2603.300c. A composition of any one of embodiments 1-290, wherein thecomposition is WV-2595.301. A composition of any one of embodiments 1-290, wherein thecomposition is not ONT-450, ONT-451, or ONT-452.302. A composition of any one of the preceding embodiments, wherein thecharacteristic sequence element comprises a mutation.303. A composition of any one of the preceding embodiments, wherein thecharacteristic sequence element is a mutation.304. A composition of any one of the preceding embodiments, wherein theoligonucleotides are at least 95% complementary to a mutant allele.305. A composition of any one of the preceding embodiments, wherein theoligonucleotides are 100% complementary to a mutant allele.306. A composition of any one of the preceding embodiments, wherein theoligonucleotides are at least 95% complementary to a target sequencecomprising a SNP, wherein the SNP is associated with a disease.307. A composition of any one of the preceding embodiments, wherein theoligonucleotides are 100% complementary to a target sequence comprisinga SNP, wherein the SNP is associated with a disease.307a. A composition of any one of the preceding embodiments, wherein theoligonucleotides selectively reduce RNA level of a mutant allele.308. A pharmaceutical composition, comprising a composition of any oneof the preceding embodiments, and a pharmaceutical carrier.308a. A composition of any one of the preceding embodiments, furthercomprising cerebrospinal fluid.309. A composition of any one of the preceding embodiments, furthercomprising artificial cerebrospinal fluid.310. A method for controlled cleavage of a nucleic acid polymer, themethod comprising steps of:

contacting a nucleic acid polymer whose nucleotide sequence comprises atarget sequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to a        target sequence found in the nucleic acid polymer;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type.        310a. A method for cleavage of a nucleic acid having a base        sequence comprising a target sequence, the method comprising        steps of:

(a) contacting a nucleic acid having a base sequence comprising a targetsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to the        target sequence in the nucleic acid;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type,        wherein the oligonucleotide targets a mutant Huntingtin gene,        and the length is from about 10 to about 50 nucleotides, wherein        the backbone linkages comprise at least one phosphorothioate,        and wherein the pattern of backbone chiral centers comprises at        least one chiral center in a Rp conformation and at least one        chiral center in a Sp conformation.        310b. A method for cleavage of a nucleic acid having a base        sequence comprising a target sequence, the method comprising        steps of:

(a) contacting a nucleic acid having a base sequence comprising a targetsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

-   -   1) a common base sequence and length, wherein the common base        sequence is or comprises a sequence that is complementary to the        target sequence in the nucleic acid;    -   2) a common pattern of backbone linkages; and    -   3) a common pattern of backbone chiral centers;        which composition is chirally controlled in that it is enriched,        relative to a substantially racemic preparation of        oligonucleotides having the particular base sequence and length,        for oligonucleotides of the particular oligonucleotide type,        wherein the oligonucleotide targets a mutant Huntingtin gene,        and the length is from about 10 to about 50 nucleotides, wherein        the backbone linkages comprise at least one phosphorothioate,        and wherein the pattern of backbone chiral centers comprises at        least one chiral center in a Rp conformation and at least one        chiral center in a Sp conformation; and

(b) cleavage of the nucleic acid mediated by a RNAseH or RNAinterference mechanism.

311. A method of embodiment 310, wherein the contacting being performedunder conditions so that cleavage of the nucleic acid polymer occurs.312. A method of any one of embodiments 310-311, wherein the cleavageoccurs with a cleavage pattern that differs from a reference cleavagepattern observed when the nucleic acid polymer is contacted undercomparable conditions with a reference oligonucleotide composition.313. A method for altering a cleavage pattern observed when a nucleicacid polymer whose nucleotide sequence includes a target sequence iscontacted with a reference oligonucleotide composition that comprisesoligonucleotides having a particular base sequence and length, whichparticular base sequence is or comprises a sequence that iscomplementary to the target sequence, the method comprising:

contacting the nucleic acid polymer with a chirally controlledoligonucleotide composition of oligonucleotides having the particularbase sequence and length, which composition is chirally controlled inthat it is enriched, relative to a substantially racemic preparation ofoligonucleotides having the particular base sequence and length, foroligonucleotides of a single oligonucleotide type characterized by:

1) the particular base sequence and length;

2) a particular pattern of backbone linkages; and

3) a particular pattern of backbone chiral centers.

314. A method of embodiment 313, wherein the contacting being performedunder conditions so that cleavage of the nucleic acid polymer occurs.315. A method of any one of embodiments 312-314, wherein the referenceoligonucleotide composition is a substantially racemic preparation ofoligonucleotides that share the common sequence and length.316. A method of any one of embodiments 312-314, wherein the referenceoligonucleotide composition is a chirally uncontrolled oligonucleotidecomposition of oligonucleotides that share the common sequence andlength.317. A method of any one of embodiments 312-316, wherein the cleavagepattern provided by the chirally controlled oligonucleotide compositiondiffers from a reference cleavage pattern in that it has fewer cleavagesites within the target sequence found in the nucleic acid polymer thanthe reference cleavage pattern.318. A method of embodiment 317, wherein the cleavage pattern providedby the chirally controlled oligonucleotide composition has a singlecleavage site within the target sequence found in the nucleic acidpolymer than the reference cleavage pattern.319. A method of embodiment 318, wherein the single cleavage site is acleavage site in the reference cleavage pattern.320. A method of embodiment 318, wherein the single cleavage site is acleavage site not in the reference cleavage pattern.321. A method of any one of embodiments 312-316, wherein the cleavagepattern provided by the chirally controlled oligonucleotide compositiondiffers from a reference cleavage pattern in that it increases cleavagepercentage at a cleavage site.322. A method of embodiment 321, wherein the cleavage site withincreased cleavage percentage is a cleavage site in the referencecleavage pattern.323. A method of embodiment 321, wherein the cleavage site withincreased cleavage percentage is a cleavage site not in the referencecleavage pattern.324. A method of any one of embodiments 310-323, wherein the chirallycontrolled oligonucleotide composition provides a higher cleavage rateof the target nucleic acid polymer than a reference oligonucleotidecomposition.325. A method of any one of embodiments 310-324, where the cleavage rateis at least 5 fold higher.326. A method of any one of embodiments 310-325, wherein the chirallycontrolled oligonucleotide composition provides a lower level ofremaining un-cleaved target nucleic acid polymer than a referenceoligonucleotide composition.327. A method of any one of embodiments 310-326, wherein the remainingun-cleaved target nucleic acid polymer is at least 5 fold lower.328. A methods of any one of embodiments 310-327, wherein the cleavageproducts from the nucleic acid polymer dissociate from oligonucleotidesof the particular oligonucleotide type in the chirally controlledoligonucleotide composition at a faster rate than from oligonucleotidesof the reference oligonucleotide composition.329. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

330. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines thetarget nucleic acid sequence, the composition being characterized inthat, when it is contacted with a system comprising transcripts of boththe target nucleic acid sequence and a similar nucleic acid sequences,transcripts of the target nucleic acid sequence are suppressed at agreater level than a level of suppression observed for a similar nucleicacid sequence.

331. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target sequence relative to its similar sequences, thecomposition being characterized in that, when it is contacted with asystem comprising transcripts of both the target allele and anotherallele of the same gene, transcripts of the particular allele aresuppressed at a level at least 2 fold greater than a level ofsuppression observed for another allele of the same gene.

332. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target sequence relative to its similar sequences, thecomposition being characterized in that, when it is contacted with asystem comprising transcripts of both the target allele and anotherallele of the same gene, transcripts of the particular allele aresuppressed at a level at least 2 fold greater than a level ofsuppression observed for another allele of the same gene.

333. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target sequence relative to its similar sequences, thecomposition being characterized in that, when it is contacted with asystem comprising transcripts of the same target nucleic acid sequence,it shows suppression of transcripts of the particular target sequence ata level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for a similar; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for a similar sequence.

334. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target sequence relative to its similar sequences, thecomposition being characterized in that, when it is contacted with asystem comprising transcripts of the same target nucleic acid sequence,it shows suppression of transcripts of the particular target sequence ata level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for a similar; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for a similar sequence.

335. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target nucleic acid sequence, the composition beingcharacterized in that, when it is contacted with a system expressingtranscripts of the target nucleic acid sequence, it shows suppression ofexpression of transcripts of the particular target nucleic acid sequenceat a level that is:

a) at least 2 fold in that transcripts from the particular targetnucleic acid sequence are detected in amounts that are 2 fold lower whenthe composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for asimilar sequence; or

c) both at least 2 fold in that transcripts from the particular targetnucleic acid sequence are detected in amounts that are 2 fold lower whenthe composition is present relative to when it is absent, and at least 2fold greater than a level of suppression observed for a similarsequence.

336. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingsteps of:contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular target nucleic acid sequence, the composition beingcharacterized in that, when it is contacted with a system expressingtranscripts of the target nucleic acid sequence, it shows suppression ofexpression of transcripts of the particular target nucleic acid sequenceat a level that is:

a) at least 2 fold in that transcripts from the particular targetnucleic acid sequence are detected in amounts that are 2 fold lower whenthe composition is present relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed for asimilar sequence; or

c) both at least 2 fold in that transcripts from the particular targetnucleic acid sequence are detected in amounts that are 2 fold lower whenthe composition is present relative to when it is absent, and at least 2fold greater than a level of suppression observed for a similarsequence.

337. A method of any one of the preceding embodiments, wherein a targetsequence is a sequence comprising a mutation, and a similar sequence isthe wild-type sequence.338. A method of any one of the preceding embodiments, wherein acharacteristic sequence element defines a particular allele of a targetsequence relative to other alleles of the same target sequence.339. A method of any one of the preceding embodiments, wherein acharacteristic sequence element defines a particular allele of a targetgene relative to other alleles of the same target gene.340. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

341. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same nucleic acid sequence, transcriptsof the particular allele are suppressed at a greater level than a levelof suppression observed for another allele of the same nucleic acidsequence.

342. A method for allele-specific suppression of a transcript from atarget gene for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

343. A method for allele-specific suppression of a transcript from atarget gene for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.

344. A method of embodiment 340 or 342, the contacting being performedunder conditions determined to permit the composition to suppresstranscripts of the particular allele.345. A method for allele-specific suppression of a transcript from atarget gene for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of both the targetallele and another allele of the same gene, transcripts of theparticular allele are suppressed at a level at least 2 fold greater thana level of suppression observed for another allele of the same gene.346. A method of embodiment 345, wherein the contacting being performedunder conditions determined to permit the composition to suppressexpression of the particular allele.347. A method of any one of embodiments 340-346, wherein transcripts ofthe particular allele are suppressed at a level at least 5, 10, 20, 50,100, 200 or 500 fold greater than a level of suppression observed foranother allele of the same gene.348. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide composition comprising oligonucleotideshaving:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

349. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising steps of:

contacting a sample comprising transcripts of the target nucleic acidsequence with a chirally controlled oligonucleotide compositioncomprising oligonucleotides of a particular oligonucleotide typecharacterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system comprising transcripts of the same targetnucleic acid sequence, it shows suppression of transcripts of theparticular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

350. A method for controlled cleavage of a nucleic acid polymer, themethod comprising contacting a nucleic acid polymer whose nucleotidesequence comprises a target sequence with an oligonucleotide or anoligonucleotide composition of any one of embodiments 540-574.351. A method for suppression of a transcript from a target nucleic acidsequence for which one or more similar nucleic acid sequences existwithin a population, each of the target and similar sequences contains aspecific nucleotide characteristic sequence element that defines thetarget sequence relative to the similar sequences, the method comprisingcontacting a sample comprising transcripts of the target nucleic acidsequence with an oligonucleotide or an oligonucleotide composition ofany one of embodiments 540-574, wherein the base sequence of theoligonucleotide is or comprises a sequence that is complementary to thecharacteristic sequence element that defines the target nucleic acidsequence.352. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising contacting a sample comprising transcripts of the targetnucleic acid sequence with an oligonucleotide or an oligonucleotidecomposition of any one of embodiments 540-574, wherein the base sequenceof the oligonucleotide is or comprises a sequence that is complementaryto the characteristic sequence element that defines a particular allele.353. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising contacting a sample comprising transcripts of the targetnucleic acid sequence with an oligonucleotide or an oligonucleotidecomposition of any one of embodiments 540-574, wherein the base sequenceof the oligonucleotide is or comprises a sequence that is complementaryto the characteristic sequence element that defines a particular allele,the oligonucleotide or oligonucleotide composition being characterizedin that, when it is contacted with a system comprising transcripts ofboth the target allele and another allele of the same gene, transcriptsof the particular allele are suppressed at a level at least 2 foldgreater than a level of suppression observed for another allele of thesame gene.354. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising contacting a sample comprising transcripts of the targetnucleic acid sequence with an oligonucleotide or an oligonucleotidecomposition of any one of embodiments 540-574, wherein the base sequenceof the oligonucleotide is or comprises a sequence that is complementaryto the characteristic sequence element that defines a particular allele,the oligonucleotide or oligonucleotide composition being characterizedin that, when it is contacted with a system expressing transcripts ofboth the target allele and another allele of the same gene, transcriptsof the particular allele are suppressed at a level at least 2 foldgreater than a level of suppression observed for another allele of thesame gene.355. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising contacting a sample comprising transcripts of the targetnucleic acid sequence with an oligonucleotide or an oligonucleotidecomposition of any one of embodiments 540-574, wherein the base sequenceof the oligonucleotide is or comprises a sequence that is complementaryto the characteristic sequence element that defines a particular allele,the oligonucleotide or oligonucleotide composition being characterizedin that, when it is contacted with a system comprising transcripts ofthe same target nucleic acid sequence, it shows suppression oftranscripts of the particular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

356. A method for allele-specific suppression of a transcript from atarget nucleic acid sequence for which a plurality of alleles existwithin a population, each of which contains a specific nucleotidecharacteristic sequence element that defines the allele relative toother alleles of the same target nucleic acid sequence, the methodcomprising contacting a sample comprising transcripts of the targetnucleic acid sequence with an oligonucleotide or an oligonucleotidecomposition of any one of embodiments 540-574, wherein the base sequenceof the oligonucleotide is or comprises a sequence that is complementaryto the characteristic sequence element that defines a particular allele,the oligonucleotide or oligonucleotide composition being characterizedin that, when it is contacted with a system expressing transcripts ofthe same target nucleic acid sequence, it shows suppression oftranscripts of the particular allele at a level that is:

a) greater than when the composition is absent;

b) greater than a level of suppression observed for another allele ofthe same nucleic acid sequence; or

c) both greater than when the composition is absent, and greater than alevel of suppression observed for another allele of the same nucleicacid sequence.

357. A method for allele-specific suppression of a transcript from atarget gene for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with anoligonucleotide composition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages;

wherein the common base sequence is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

358. A method for allele-specific suppression of a transcript from atarget gene for which a plurality of alleles exist within a population,each of which contains a specific nucleotide characteristic sequenceelement that defines the allele relative to other alleles of the sametarget gene, the method comprising steps of:

contacting a sample comprising transcripts of the target gene with achirally controlled oligonucleotide composition comprisingoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages;

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type;

wherein the common base sequence for the oligonucleotides of theparticular oligonucleotide type is or comprises a sequence that iscomplementary to the characteristic sequence element that defines aparticular allele, the composition being characterized in that, when itis contacted with a system expressing transcripts of the target gene, itshows suppression of expression of transcripts of the particular alleleat a level that is:

a) at least 2 fold in that transcripts from the particular allele aredetected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent;

b) at least 2 fold greater than a level of suppression observed foranother allele of the same gene; or

c) both at least 2 fold in that transcripts from the particular alleleare detected in amounts that are 2 fold lower when the composition ispresent relative to when it is absent, and at least 2 fold greater thana level of suppression observed for another allele of the same gene.

359. A method of any one of the preceding embodiments, whereintranscripts from the particular allele are detected in amounts that are2 fold or more when the composition is absent relative to when it ispresent.360. A method of any one of the preceding embodiments, wherein the levelof transcripts of another allele of the same gene is at least 2 foldgreater than the level of transcripts of the particular allele.361. A method of any one of the preceding embodiments, whereintranscripts from the particular allele are detected in amounts that are2 fold or more when the composition is absent relative to when it ispresent, and the level of transcripts of another allele of the same geneis at least 2 fold greater than the level of transcripts of theparticular allele.362. A method of any one of the preceding embodiments, the contactingbeing performed under conditions determined to permit the composition tosuppress transcripts of the particular allele.363. A method of any one of the preceding embodiments, wherein thecontacting being performed under conditions determined to permit thecomposition to suppress expression of the particular allele.364. A method of any one of the preceding embodiments, whereintranscripts of the particular allele are suppressed at a level that isat least 5, 10, 20, 50, 100, 200 or 500 fold in that transcripts fromthe particular allele are detected in amounts that are 2 fold lower whenthe composition is present relative to when it is absent.365. A method of any one of the preceding embodiments, whereintranscripts of the particular allele are suppressed at a level that isat least 5, 10, 20, 50, 100, 200 or 500 fold greater than a level ofsuppression observed for another allele of the same gene.366. A method of any one of the preceding embodiments, wherein thesystem is an in vitro or in vivo system.366a A method of any one of the preceding embodiments, wherein themethod is performed in vitro or in vivo.367. A method of any one of the preceding embodiments, wherein thesystem comprises one or more cells, tissues or organs.368. A method of any one of the preceding embodiments, wherein thesystem comprises one or more organisms.369. A method of any one of the preceding embodiments, wherein thesystem comprises one or more subjects.370. A method of any one of the preceding embodiments, whereintranscripts of the particular allele are cleaved.371. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element is present within anintron of the target nucleic acid sequence or gene.372. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element is present within anexon of the target nucleic acid sequence or gene.373. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element spans an exon and anintron of the target nucleic acid sequence or gene.374. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element comprises amutation.375. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element is a mutation.376. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element comprises a SNP.377. A method of any one of the preceding embodiments, wherein thespecific nucleotide characteristic sequence element is a SNP.378. A method of any one of the preceding embodiments, wherein theoligonucleotide composition is administered to a subject.379. A method of any one of the preceding embodiments, wherein thetarget nucleic acid polymer or transcripts are oligonucleotides.380. A method of any one of the preceding embodiments, wherein thetarget nucleic acid polymer or transcripts are RNA.381. A method of any one of the preceding embodiments, wherein thetarget nucleic acid polymer or transcripts are newly transcribed RNA.382. A method of any one of the preceding embodiments, whereinoligonucleotides of the particular oligonucleotide type in the chirallycontrolled oligonucleotide composition form duplexes with the nucleicacid polymer or transcripts.383. A method of any one of the preceding embodiments, wherein thenucleic acid polymer or transcripts are cleaved by an enzyme.384. A method of any one of the preceding embodiments, wherein theenzyme is RNase H.385. A method of any one of the preceding embodiments, wherein the SNPis a SNP related to Huntington's disease.386. A method of any one of the preceding embodiments, wherein the SNPis a SNP found in the Huntingtin gene.387. A method of any one of the preceding embodiments, wherein the SNPis selected from rs362307, rs7685686, rs362268, or rs362306.388. A method of embodiments 310-387, wherein the SNP is rs362307.389. A method of embodiments 310-387, wherein the single nucleotidepolymorphism is rs7685686.390. A method of embodiments 310-387, wherein the single nucleotidepolymorphism is rs362268.391. A method of embodiments 310-387, wherein the single nucleotidepolymorphism is rs362306.392. A method of embodiments 310-391, wherein position 11 of theoligonucleotides as counted from the 5′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.393. A method of embodiments 310-391, wherein position 12 of theoligonucleotides as counted from the 5′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.394. A method of embodiments 310-391, wherein position 13 of theoligonucleotides as counted from the 5′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.395. A method of embodiments 310-391, wherein position 8 of theoligonucleotides as counted from the 3′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.396. A method of embodiments 310-391, wherein position 9 of theoligonucleotides as counted from the 3′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.397. A method of embodiments 310-391, wherein position 10 of theoligonucleotides as counted from the 3′-terminus of the oligonucleotidesaligns with a single nucleotide polymorphism.398. A method of any one of the preceding embodiments, wherein theoligonucleotides comprise one or more wing regions and a common coreregion, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more chiral internucleotidic linkages.

399. A method of any one of embodiments 310-398, wherein position 6 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.400. A method of any one of embodiments 310-398, wherein position 7 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.401. A method of any one of embodiments 310-398, wherein position 8 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.402. A method of any one of embodiments 310-398, wherein position 3 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.403. A method of any one of embodiments 310-398, wherein position 4 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.404. A method of any one of embodiments 310-398, wherein position 5 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.405. A method of any one of the preceding embodiments, wherein:

each wing region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages andone or more natural phosphate linkage; and

the core region independently has a length of two or more bases, whereineach internucleotidic linkage in the core region is chiral, only one ofinternucleotidic linkage in the core region is Rp, and each of the otherinternucleotidic linkages in the core region is Sp.

406. A method of any one of the preceding embodiments, wherein theoligonucleotides are hemimers having the structure of wing-core.407. A method of any one of embodiments 310-405, wherein theoligonucleotides are hemimers having the structure of core-wing.408. A method of any one of embodiments 310-405, wherein theoligonucleotides are gapmers having the structure of wing-core-wing.409. A method of any one of the preceding embodiments, wherein level oftranscripts from a disease-causing allele is selectively suppressed.410. A method of any one of the preceding embodiments, wherein level ofa protein translated from transcripts from a disease-causing allele aresuppressed.411. A method for treating or preventing Huntington's Disease in asubject, comprising administering to the subject an oligonucleotidecomposition comprising oligonucleotides having:

1) a common base sequence and length; and

2) a common pattern of backbone linkages.

412. A method for treating or preventing Huntington's Disease in asubject, comprising administering to the subject a chirally controlledoligonucleotide composition comprising oligonucleotides of a particularoligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.413. A method of embodiment 411 or 412, wherein the oligonucleotidescomprise one or more wing regions and a common core region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more chiral internucleotidic linkages.

414. A method of any one of embodiments 411-4113, wherein:

each wing region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages andone or more natural phosphate linkage; and

the core region independently has a length of two or more bases, whereineach internucleotidic linkage in the core region is chiral, only one ofinternucleotidic linkage in the core region is Rp, and each of the otherinternucleotidic linkages in the core region is Sp.

415. A method of any one of embodiments 411-414, wherein theoligonucleotides are hemimers having the structure of wing-core.416. A method of any one of embodiments 411-414, wherein theoligonucleotides are hemimers having the structure of core-wing.417. A method of any one of embodiments 411-414, wherein theoligonucleotides are gapmers having the structure of wing-core-wing.418. A method of any one of embodiments 411-417, wherein position 11 ofthe oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.419. A method of any one of embodiments 411-417, wherein position 12 ofthe oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.420. A method of any one of embodiments 411-417, wherein position 13 ofthe oligonucleotides as counted from the 5′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.421. A method of any one of embodiments 411-417, wherein position 8 ofthe oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.422. A method of any one of embodiments 411-417, wherein position 9 ofthe oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.423. A method of any one of embodiments 411-417, wherein position 10 ofthe oligonucleotides as counted from the 3′-terminus of theoligonucleotides aligns with a single nucleotide polymorphism.424. A method of any one of embodiments 411-417, wherein position 6 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.425. A method of any one of embodiments 411-417, wherein position 7 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.426. A method of any one of embodiments 411-417, wherein position 8 ofthe core region as counted from the 5′-terminus of the core regionaligns with a single nucleotide polymorphism.427. A method of any one of embodiments 411-417, wherein position 3 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.428. A method of any one of embodiments 411-417, wherein position 4 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.429. A method of any one of embodiments 411-417, wherein position 5 ofthe core region as counted from the 3′-terminus of the core regionaligns with a single nucleotide polymorphism.430. A method of any one of embodiments 411-429, wherein the methodameliorating a symptom of Huntington's Disease.431. A method of any one of embodiments 411-429, wherein the methodslowing onset of Huntington's Disease.432. A method of any one of embodiments 411-429, wherein the methodslowing progression of Huntington's Disease.433. A method of any one of embodiments 411-432, wherein the subject hasa SNP related to Huntington's Disease.434. A method of any one of embodiments 411-433, wherein the subject hasa SNP in the subject's Huntingtin gene.435. A method of any one of embodiments 411-434, wherein the subject hasa SNP, wherein one allele is mutant Huntingtin associated with expandedCAG repeats.436. A method of any one of embodiments 411-435, wherein the subject hasa SNP selected from rs362307, rs7685686, rs362268, rs2530595, rs362331,or rs362306.436a. A method of any one of embodiments 411-435, wherein the subjecthas a SNP selected from rs362307, rs7685686, rs362268, or rs362306.437. A method of any one of embodiments 411-436, wherein the subject hasthe SNP rs362307.438. A method of any one of embodiments 411-436, wherein the subject hasthe SNP rs7685686.439. A method of any one of embodiments 411-436, wherein the subject hasthe SNP rs362268.440. A method of any one of embodiments 411-436, wherein the subject hasthe SNP rs362306.440a. A method of any one of embodiments 411-436, wherein the subjecthas the SNP rs2530595.440b. A method of any one of embodiments 411-436, wherein the subjecthas the SNP rs362331.441. A composition of any of embodiments 1-309, wherein a substantiallyracemic preparation of oligonucleotides is prepared bynon-stereoselective preparation.442. A composition of any one of embodiments 1-309 and 441, wherein asubstantially racemic preparation of oligonucleotides is prepared bynon-stereoselective preparation, wherein a chiral auxiliary is not usedfor formation of a chiral internucleotidic linkage.443. A composition of any one of embodiments 1-309 and 441-442, whereina substantially racemic preparation of oligonucleotides is prepared bynon-stereoselective preparation, wherein at least one chiralinternucleotidic linkage is formed with less than 80:20 diastereomericselectivity.444. A composition of any one of embodiments 1-309 and 441-443, whereina substantially racemic preparation of oligonucleotides is prepared bynon-stereoselective preparation, wherein at least one chiralinternucleotidic linkage is formed with less than 90:10 diastereomericselectivity.445. A composition of any one of embodiments 1-309 and 441-444, whereina substantially racemic preparation of oligonucleotides is prepared bynon-stereoselective preparation, wherein at least one chiralinternucleotidic linkage is formed with less than 95:5 diastereomericselectivity.446. A composition of any one of embodiments 1-309 and 441-445, whereina substantially racemic preparation of oligonucleotides is prepared bynon-stereoselective preparation, wherein at least one chiralinternucleotidic linkage is formed with less than 97:3 diastereomericselectivity.447. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 90:10diastereomeric selectivity.448. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 95:5 diastereomericselectivity.449. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 96:4 diastereomericselectivity.450. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 97:3 diastereomericselectivity.451. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 98:2 diastereomericselectivity.452. A composition of any one of embodiments 1-309, wherein each chiralinternucleotidic linkage is formed with greater than 98:2 diastereomericselectivity.453. A composition of any one of embodiments 443-452, wherein thediastereomeric selectivity for forming a chiral internucleotidic linkageis measured by forming a dimeric oligonucleotide comprising the chiralinternucleotidic linkage and the nucleosides to both sides of the chiralinternucleotidic linkage under the same or comparable reactionconditions.454. A method for preparing an oligonucleotide composition for selectivesuppression of a transcript of a target nucleic acid sequence,comprising providing an oligonucleotide composition comprising apredetermined level of oligonucleotides of a particular oligonucleotidetype characterized by:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which pattern comprises(Sp)_(m)(Rp)_(n), (Rp)_(n)(Sp)_(m), (Np)_(t)(Rp)_(n)(Sp)_(m), or(Sp)_(t)(Rp)_(n)(Sp)_(m), wherein:

m is 1-50;

n is 1-10;

t is 1-50;

each Np is independently Rp or Sp;

wherein the target nucleic acid sequence comprises a characteristicsequence element that defines the target nucleic acid sequence from asimilar nucleic acid sequence;

wherein the common base sequence is a sequence whose DNA cleavagepattern and/or stereorandom cleavage pattern has a cleavage site withinor in the vicinity of the target nucleic acid sequence.

455. A method of embodiment 454, wherein the pattern comprises(Sp)_(m)(Rp)_(n).456. A method of embodiment 454, wherein the pattern comprises(Rp)_(n)(Sp)_(m).457. A method of embodiment 454, wherein the pattern comprises(Np)_(t)(Rp)_(n)(Sp)_(m).458. A method of embodiment 454, wherein the pattern comprises(Sp)_(t)(Rp)_(n)(Sp)_(m).459. A method of embodiment 454, wherein the pattern is a pattern in anyone of embodiments 145-157.460. A method of any one of embodiments 454-458, wherein a cleavage siteis in any one of embodiments 247-285.461. A method of any one of the preceding embodiments, wherein theoligonucleotide composition is a composition of any one of embodiments1-309 and 441-453.462. A composition or method of any one of the preceding embodiments,wherein the sequence of the oligonucleotide in a chirally controlledoligonucleotide composition comprises, consists of, or is the sequenceof any oligonucleotide described herein, or selected from Tables N1A,N2A, N3A, N4A or 8; or WV-1092, WVE120101, WV-2603 or WV-2595.463. A composition comprising a lipid and an oligonucleotide.463a. The composition of any one of the preceding embodiments, whereinthe composition comprises one or more lipids conjugated with one or moreoligonucleotides in the composition.464. A composition comprising an oligonucleotide and a lipid selectedfrom the list of: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl.464a. A composition comprising an oligonucleotide and a lipid selectedfrom the list of: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, and dilinoleyl.465. A composition comprising an oligonucleotide and a lipid selectedfrom:

466. A composition comprising an oligonucleotide and a lipid, whereinthe lipid comprises a C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic group.467. An oligonucleotide composition comprising a plurality ofoligonucleotides, which share:1) a common base sequence;2) a common pattern of backbone linkages; and3) a common pattern of backbone phosphorus modifications; wherein one ormore oligonucleotides of the plurality are individually conjugated to alipid.468. A chirally controlled oligonucleotide composition comprising aplurality of oligonucleotides, which share:1) a common base sequence;2) a common pattern of backbone linkages; and3) a common pattern of backbone phosphorus modifications;wherein:the composition is chirally controlled in that the plurality ofoligonucleotides share the same stereochemistry at one or more chiralinternucleotidic linkages;one or more oligonucleotides of the plurality are individuallyconjugated to a lipid; and one or more oligonucleotides of the pluralityare optionally and individually conjugated to a targeting compound ormoiety.469. A method of delivering an oligonucleotide to a cell or tissue in ahuman subject, comprising:(a) providing a composition of any one of the preceding embodiments; and(b) Administering the composition to the human subject such that theoligonucleotide is delivered to a cell or tissue in the subject.470. A method for delivering an oligonucleotide to a cell or tissuecomprising preparing a composition according to any one of the precedingembodiments and treating [contacting] the cell or tissue with thecomposition.471. A method of modulating the level of a transcript or gene product ofa gene in a cell, the method comprising the step of contacting the cellwith a composition according to any one of the preceding embodiments,wherein the oligonucleotide is capable of modulating the level of thetranscript or gene product.472. A method for inhibiting expression of a gene in a cell or tissuecomprising preparing a composition according to any one of the precedingembodiments and treating the cell or tissue with the composition.473. A method for inhibiting expression of a gene in a cell or tissue ina mammal comprising preparing a composition according to any one of thepreceding embodiments and administering the composition to the mammal.474. A method of treating a disease that is caused by theover-expression of one or several proteins in a cell or tissue in asubject, said method comprising the administration of a compositionaccording to any one of the preceding embodiments to the subject.475. A method of treating a disease that is caused by a reduced,suppressed or missing expression of one or several proteins in asubject, said method comprising the administration of a compositionaccording to any one of the preceding embodiments to the subject.476. A method for generating an immune response in a subject, saidmethod comprising the administration of a composition according to anyone of the preceding embodiments to the subject, wherein thebiologically active compound is an immunomodulating nucleic acid.477. A method for treating a sign and/or symptom of Huntington's Diseaseby providing a composition of any one of the preceding embodiments andadministering the composition to the subject.478. A method of modulating the amount of RNaseH-mediated cleavage in acell, the method comprising the step of contacting the cell with acomposition according to any one of the preceding embodiments, whereinthe oligonucleotide is capable of modulating the amount ofRNaseH-mediated cleavage.479. A method of administering an oligonucleotide to a subject in needthereof, comprising steps of providing a composition comprising theagent a lipid, and administering the composition to the subject, whereinthe agent is any agent disclosed herein, and wherein the lipid is anylipid disclosed herein.480. A method of treating a disease in a subject, the method comprisingsteps of providing a composition comprising the agent a lipid, andadministering a therapeutically effective amount of the composition tothe subject, wherein the agent is any agent disclosed herein, andwherein the lipid is any lipid disclosed herein, and wherein the diseaseis any disease disclosed herein.481. The composition or method of any one of the preceding embodiments,wherein a lipid comprises an optionally substituted C₁₀-C₄₀ saturated orpartially unsaturated aliphatic chain.482. The composition or method of any one of the preceding embodiments,wherein a lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.483. The composition or method of any one of the preceding embodiments,wherein a lipid comprises a C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic group.484. The composition or method of any one of the preceding embodiments,wherein a lipid comprises an unsubstituted C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain.485. The composition or method of any one of the preceding embodiments,wherein a lipid comprises no more than one optionally substitutedC₁₀-C₄₀ linear, saturated or partially unsaturated, aliphatic chain.486. The composition or method of any one of the preceding embodiments,wherein a lipid comprises two or more optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain.487. The composition or method of any one of the preceding embodiments,wherein a lipid comprises no tricyclic or polycyclic moiety.488. The composition or method of any one of the preceding embodiments,wherein a lipid has the structure of R¹—COOH, wherein R¹ is anoptionally substituted C₁₀-C₄₀ saturated or partially unsaturatedaliphatic chain.489. The composition or method of any one of embodiment 16, wherein thelipid is conjugated through its carboxyl group.490. The composition or method according to any one of the precedingembodiments, wherein the lipid is selected from:

491. The composition or method of any one of the preceding embodiments,wherein the lipid is conjugated to the oligonucleotide.492. The composition or method of any one of the preceding embodiments,wherein the lipid is directly conjugated to the oligonucleotide.493. The composition or method of any one of the preceding embodiments,wherein the lipid is conjugated to the oligonucleotide via a linker.494. The composition or method of any one of the preceding embodiments,wherein the linker is selected from: an uncharged linker; a chargedlinker; a linker comprising an alkyl; a linker comprising a phosphate; abranched linker; an unbranched linker; a linker comprising at least onecleavage group; a linker comprising at least one redox cleavage group; alinker comprising at least one phosphate-based cleavage group; a linkercomprising at least one acid-cleavage group; a linker comprising atleast one ester-based cleavage group; and a linker comprising at leastone peptide-based cleavage group.495. The composition or method of any one of the preceding embodiments,wherein each oligonucleotide of the plurality is individually conjugatedto the same lipid at the same location.496. The composition or method of any one of the preceding embodiments,wherein a lipid is conjugated to an oligonucleotide through a linker.497. The composition or method of any one of the preceding embodiments,wherein one or more oligonucleotides of the plurality are independentlyconjugated to a targeting compound or moiety.498. The composition or method of any one of the preceding embodiments,wherein one or more oligonucleotides of the plurality are independentlyconjugated to a lipid and a targeting compound or moiety.499. The composition or method of any one of the preceding embodiments,wherein one or more oligonucleotides of the plurality are independentlyconjugated to a lipid at one end and a targeting compound or moiety atthe other.500. The composition or method of any one of the preceding embodiments,wherein oligonucleotides of the plurality share the same chemicalmodification patterns.501. The composition or method of any one of the preceding embodiments,wherein oligonucleotides of the plurality share the same chemicalmodification patterns comprising one or more base modifications.502. The composition or method of any one of the preceding embodiments,wherein oligonucleotides of the plurality share the same chemicalmodification patterns comprising one or more sugar modifications.503. The composition or method of any one of the preceding embodiments,wherein the common base sequence is capable of hybridizing with atranscript in a cell, which transcript contains a mutation that islinked to a muscle disease, or whose level, activity and/or distributionis linked to a muscle disease.504. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide is a nucleic acid.505. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide is an oligonucleotide.506. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide is an oligonucleotide which mediates exonskipping.507. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide is a stereodefined oligonucleotide whichmediates exon skipping.508. The composition or method of any one of the preceding embodiments,wherein the disease or disorder is a muscle-related disease or disorder.509. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted, C₁₀-C₈₀ saturatedor partially unsaturated aliphatic group, wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡—,a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein eachvariable is independently as defined and described herein.510. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₈₀ saturatedor partially unsaturated, aliphatic chain.511. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain.512. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₆₀ saturatedor partially unsaturated, aliphatic chain.513. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.514. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₄₀ saturatedor partially unsaturated, aliphatic chain.515. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.516. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted, C₁₀-C₆₀ saturatedor partially unsaturated aliphatic group, wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein eachvariable is independently as defined and described herein.517. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₈₀ saturatedor partially unsaturated, aliphatic chain.518. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.519. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.520. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted, C₁₀-C₄₀ saturatedor partially unsaturated aliphatic group, wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein eachvariable is independently as defined and described herein.521. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₄₀ saturatedor partially unsaturated, aliphatic chain.522. The composition or method of any one of the preceding embodiments,wherein the lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.523. The composition or method of any one of the preceding embodiments,wherein the composition further comprises one or more additionalcomponents selected from: a polynucleotide, carbonic anhydraseinhibitor, a dye, an intercalating agent, an acridine, a cross-linker,psoralene, mitomycin C, a porphyrin, TPPC4, texaphyrin, Sapphyrin, apolycyclic aromatic hydrocarbon phenazine, dihydrophenazine, anartificial endonuclease, a chelating agent, EDTA, an alkylating agent, aphosphate, an amino, a mercapto, a PEG, PEG-40K, MPEG, [MPEG]₂, apolyamino, an alkyl, a substituted alkyl, a radiolabeled marker, anenzyme, a hapten biotin, a transport/absorption facilitator, aspirin,vitamin E, folic acid, a synthetic ribonuclease, a protein, aglycoprotein, a peptide, a molecule having a specific affinity for aco-ligand, an antibody, a hormone, a hormone receptor, a non-peptidicspecies, a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, or adrug.524. The composition or method of any one of the preceding embodiments,wherein the lipid comprises a C₁₀-C₈₀ linear, saturated or partiallyunsaturated, aliphatic chain.525. The composition or method of any one of the preceding embodiments,wherein the composition further comprises a linker linking theoligonucleotide and the lipid, wherein the linker is selected from: anuncharged linker; a charged linker; a linker comprising an alkyl; alinker comprising a phosphate; a branched linker; an unbranched linker;a linker comprising at least one cleavage group; a linker comprising atleast one redox cleavage group; a linker comprising at least onephosphate-based cleavage group; a linker comprising at least oneacid-cleavage group; a linker comprising at least one ester-basedcleavage group; a linker comprising at least one peptide-based cleavagegroup.526. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition.527. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of any oligonucleotide describedherein.528. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of any oligonucleotide listed inTable 4.529. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of a splice-switchingoligonucleotide.530. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of an oligonucleotide capable ofskipping or mediating skipping of an exon in the dystrophin gene.531. The composition or method of any of the preceding embodiments,wherein the oligonucleotide is a chirally controlled oligonucleotidecomposition.532. The composition or method of any of the preceding embodiments,wherein the disease or disorder is Huntington's Disease.533. The composition or method of any of the preceding embodiments,wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.534. The composition or method of any of the preceding embodiments,wherein the oligonucleotide comprises, consists of or is the sequence ofany oligonucleotide disclosed herein.535. The composition or method of any of the preceding embodiments,wherein the oligonucleotide is capable of differentiating between awild-type and a mutant Huntingtin allele.536. The composition or method of any of the preceding embodiments,wherein the oligonucleotide is capable of participating inRNaseH-mediated cleavage of a mutant Huntingtin gene mRNA.537. The composition or method of any of the preceding embodiments,wherein the oligonucleotide comprises, consists of or is the sequence ofany oligonucleotide disclosed in Table 4.538. The composition or method of any one of the preceding embodiments,wherein the oligonucleotide comprises or consists of or is anoligonucleotide or oligonucleotide composition or chirally controlledoligonucleotide composition, wherein the sequence of the oligonucleotidecomprises or consists of the sequence of any of: WV-1092, WV-2595, orWV-2603.539. The composition or method of any one of the preceding embodiments,wherein the sequence of an oligonucleotide includes any one or more of:base sequence (including length); pattern of chemical modifications tosugar and base moieties; pattern of backbone linkages; pattern ofnatural phosphate linkages, phosphorothioate linkages, phosphorothioatetriester linkages, and combinations thereof; pattern of backbone chiralcenters; pattern of stereochemistry (Rp/Sp) of chiral internucleotidiclinkages; pattern of backbone phosphorus modifications; pattern ofmodifications on the internucleotidic phosphorus atom, such as —S—, and-L-R¹ of formula I.540. An oligonucleotide comprising a sequence that shares greater thanabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% identity with a sequencefound in a provided example oligonucleotide.540a. The oligonucleotide of embodiment 540, wherein the sequence of theoligonucleotide is the sequence of a provided example oligonucleotide.540b. The oligonucleotide of embodiment 540 or 540a, wherein theprovided example oligonucleotide is an oligonucleotide selected fromTable N1A, N2A, N3A, N4A or 8.541. The oligonucleotide of embodiment 540, wherein the provided exampleoligonucleotide is WV-1092.542. The oligonucleotide of embodiment 540, wherein the provided exampleoligonucleotide is WV-2595.543. The oligonucleotide of embodiment 540, wherein the provided exampleoligonucleotide is WV-2603544. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises the sequence found in the providedexample oligonucleotide.545. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide consists of the sequence found in theprovided example oligonucleotide.546. An oligonucleotide of any one of the preceding embodiments, whereinthe oligonucleotide comprises one or more natural phosphate linkages andone or more modified internucleotidic linkages.547. The oligonucleotide of embodiment 546, wherein the oligonucleotideis an oligonucleotide of any one of embodiments 540-545.548. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore natural phosphate linkages.549. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises one or more modifiedinternucleotidic linkages.550. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises two or more modifiedinternucleotidic linkages.551. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more modified internucleotidiclinkages.552. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more modified internucleotidic linkages.553. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide comprises 10 or more modifiedinternucleotidic linkages.554. The oligonucleotide of any one of the preceding embodiments,wherein at least one of the modified internucleotidic linkages is achirally controlled internucleotidic linkage in that oligonucleotideshaving the same sequence and chemical modifications within a compositionshare the same configuration, either Rp or Sp, at the chiral phosphorusatom of the modified internucleotidic linkage.555. The oligonucleotide of any one of the preceding embodiments,wherein at least two modified internucleotidic linkages are chirallycontrolled.556. The oligonucleotide of any one of the preceding embodiments,wherein at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 modified internucleotidic linkages are chirally controlled.557. The oligonucleotide of any one of the preceding embodiments,wherein at least one modified internucleotidic linkage within aconsecutive modified internucleotidic linkage region is chirallycontrolled.558. The oligonucleotide of any one of the preceding embodiments,wherein at least two modified internucleotidic linkages within aconsecutive modified internucleotidic linkage region are chirallycontrolled.559. The oligonucleotide of any one of the preceding embodiments,wherein at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 modified internucleotidic linkages within a consecutivemodified internucleotidic linkage region are chirally controlled.560. The oligonucleotide of any one of the preceding embodiments,wherein each modified internucleotidic linkage within a consecutivemodified internucleotidic linkage region is chirally controlled.561. The oligonucleotide of any one of the preceding embodiments,wherein each modified internucleotidic linkage is chirally controlled.562. The oligonucleotide of any one of the preceding embodiments,wherein a provided oligonucleotide comprises a (Sp)xRp(Sp)y pattern,wherein each of x and y is independently 1-20, and the sum of x and y is1-50.563. The oligonucleotide of any one of the preceding embodiments,wherein each of x and y is independently 2-20.564. The oligonucleotide of any one of the preceding embodiments,wherein at least one of x and y is greater than 5, 6, 7, 8, 9, or 10.565. The oligonucleotide of any one of the preceding embodiments,wherein the sum of x and y is greater than 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20.566. The oligonucleotide of any one of the preceding embodiments,wherein a provided oligonucleotide comprises one or more chemicalmodifications.567. The oligonucleotide of any one of the preceding embodiments,wherein a provided oligonucleotide comprises one or more basemodifications.568. The oligonucleotide of any one of the preceding embodiments,wherein a provided oligonucleotide comprises one or more sugarmodifications.569. The oligonucleotide of any one of the preceding embodiments,wherein a sugar modification is a 2′-modification.570. The oligonucleotide of any one of the preceding embodiments,wherein a sugar modification is LNA.571. The oligonucleotide of any one of the preceding embodiments,wherein a provided oligonucleotide is a chirally controlledoligonucleotide.572. The oligonucleotide of any one of the preceding embodiments,wherein the oligonucleotide is conjugated to a targeting component.573. A oligonucleotide composition, comprising an oligonucleotide of anyone of the preceding embodiments.573a. The composition of embodiment 573, wherein the composition is achirally controlled oligonucleotide composition comprising apredetermined level of the oligonucleotide.574. The composition of any one of the preceding embodiments, or thecomposition in the method of any one of the preceding embodiments,further comprising a selectivity agent selected from: the group ofcompounds which binds specifically to one or more neurotransmittertransporters selected from the group consisting of a dopaminetransporter (DAT), a serotonin transporter (SERT), and a norepinephrinetransporter (NET); the group consisting of a dopamine reuptake inhibitor(DRI), a selective serotonin reuptake inhibitor (SSRI), a noradrenalinereuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor(NDRI), and a serotonin-norepinephrine-dopamine reuptake inhibitor(SNDRI); the group consisting of a triple reuptake inhibitor, anoradrenaline dopamine double reuptake inhibitor, a serotonin singlereuptake inhibitor, a noradrenaline single reuptake inhibitor, and adopamine single reuptake inhibitor; and the group consisting of adopamine reuptake inhibitor (DRI), a Norepinephrine-Dopamine ReuptakeInhibitor (NDRI) and a serotonin-Norepinephrine-Dopamine ReuptakeInhibitor (SNDRI).575. A method for treating or preventing Huntington's Disease in asubject, comprising administering to the subject an oligonucleotide or acomposition of any one of the preceding embodiments.575a. A method of any one of the preceding embodiments, wherein theoligonucleotide or composition is administered via intrathecaladministration.576. A method for preparing an oligonucleotide, comprising providing achiral reagent having the structure of Formula 3-AA.577. A method for preparing an oligonucleotide, comprising providing achiral reagent having the structure of

578. The method of any one of the preceding embodiments, wherein thechiral reagent is chirally pure.579. A method for preparing an oligonucleotide, comprising providing acompound comprising a moiety from a chiral reagent having the structureof any one of the preceding embodiments, wherein —W¹H and —W²H, or thehydroxyl and amino groups, form bonds with the phosphorus atom of thephosphoramidite.580. The method of embodiment 579, wherein the compound has thestructure of

581. The method of embodiment 580, wherein R connected to the 5′-O is ahydroxyl protecting group.582. The method of embodiment 581, wherein the hydroxyl protecting groupis DMTr.583. The method of embodiment 582, wherein B^(PRO) is a protectednucleobase.584. The method of embodiment 583, wherein the nucleobase is anoptionally substituted nucleobase selected from A, T, C and G.585. The method of any one of the preceding embodiments, wherein W¹ is—NG⁵, W² is O.586. The method of any one of the preceding embodiments, wherein each ofG¹ and G³ is independently hydrogen or an optionally substituted groupselected from C₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl, G² is—C(R)₂Si(R)₃, and G⁴ and G⁵ are taken together to form an optionallysubstituted saturated, partially unsaturated or unsaturatedheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused.587. The method of any one of the preceding embodiments, wherein each Ris independently hydrogen, or an optionally substituted group selectedfrom C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl.588. The method of any one of the preceding embodiments, wherein G¹ ishydrogen.589. The method of any one of the preceding embodiments, wherein G² is—C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, and eachR of —Si(R)₃ is independently an optionally substituted group selectedfrom C₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl.590. The method of any one of the preceding embodiments, wherein atleast one R of —Si(R)₃ is independently optionally substituted C₁₋₁₀alkyl.591. The method of any one of the preceding embodiments, wherein atleast one R of —Si(R)₃ is independently optionally substituted phenyl.592. The method of any one of the preceding embodiments, wherein one Rof —Si(R)₃ is independently optionally substituted C₁₋₁₀ alkyl, and eachof the other two R is independently optionally substituted phenyl.593. The method of any one of the preceding embodiments, wherein G² isoptionally substituted —CH₂Si(Me)(Ph)₂.594. The method of any one of the preceding embodiments, wherein G² is—CH₂Si(Me)(Ph)₂.595. The method of any one of the preceding embodiments, wherein G³ ishydrogen.596. The method of any one of the preceding embodiments, wherein G⁴ andG⁵ are taken together to form an optionally substituted saturated 5-6membered ring containing one nitrogen atom.597. The method of any one of the preceding embodiments, wherein G⁴ andG⁵ are taken together to form an optionally substituted saturated5-membered ring containing one nitrogen atom.598. The method of any one of the preceding embodiments, wherein thechiral reagent is

599. The method of any one of the preceding embodiments, comprisingproviding a fluoro-containing reagent.600. The method of any one of the preceding embodiments, wherein thefluoro-containing reagent is TBAF.601. The method of any one of the preceding embodiments, comprisingusing a linker that is stable to a TBAF condition for removing thechiral reagent.602. The method of any one of the preceding embodiments, wherein thelinker is an SP linker.603. The method of any one of embodiments 576-599, wherein thefluoro-containing reagent is HF—NR₃.604. The method of embodiment 603, wherein the fluoro-containing reagentis HF-NEt₃.605. The method of embodiment 603 or 604, comprising using a linker thatis stable to a HF—NR₃ condition for removing the chiral reagent.606. The method of any one of embodiments 603-605, wherein the linker isa succinyl linker.

EXAMPLES

The foregoing has been a description of certain non-limiting embodimentsof the disclosure. Accordingly, it is to be understood that theembodiments of the disclosure herein described are merely illustrativeof the application of the principles of the disclosure. Reference hereinto details of the illustrated embodiments is not intended to limit thescope of the claims.

Example 1. In Vitro Metabolic Stabilities of Human Chiromersens inPreincubated Rat Whole Liver Homogenates

The present Example describes comparisons of in vitro whole rat liverhomogenate stability of Mipomersen (stereochemical mixture) withchirally controlled oligonucleotide compositions of Mipomersen(“chiromersens”). The method, among other things, is useful in screeningcompounds to predict in vivo half lives.

As is known in the art, Mipomersen (previously ISIS 301012, sold underthe trade name Kynamro) is a 20mer oligonucleotide whose base sequenceis antisense to a portion of the apolipoprotein B gene. Mipomerseninhibits apolipoprotein B gene expression, presumably by targeting mRNA.Mipomersen has the following structure:

(SEQ ID NO: 43) G*-C*-C*-U*-C*-dA-dG-dT-dC-dT-dG-dmC-dT-dT-dmC-G*-C*-A*-C*-C* [d = 2′-deoxy, *= 2′-O-(2-methoxyethyl)]with 3′→5′ phosphorothioate linkages. Thus, Mipomersen has2′-O-methoxyethyl-modified ribose residues at both ends, and deoxyriboseresidues in the middle.

Tested chirally pure Mipomersen analogs described in this Exampleincluded 3′→5′ phosphorothioate linkages. In some embodiments, testedanalogs include one or more 2′-O-(2-methoxyethyl)-modified residues; insome embodiments, tested analogs include only 2′-deoxy residues.Particular tested analogs had the structures set forth below in Tables 3and 4.

Protocol:

We used the protocol reported by Geary et al. (Oligonucleotides, Volume20, Number 6, 2010) with some modifications.

Test System:

Six male Sprague-Dawley rats (Rattus norvegicus) were supplied byCharles River Laboratories, Inc., (Hollister, Calif.), and were receivedat SNBL USA.

Tissue Collection:

Animals were acclimated to the study room for two days prior to tissuecollection. At the time of tissue collection, animals were anesthetizedwith an intraperitoneal (IP) injection of sodium pentobarbital solution.Liver perfusion was performed using 500 mL of chilled saline/animal,administered via the hepatic portal vein. After perfusion, the liverswere dissected and maintained on ice. Livers were minced into smallpieces then weighed.

Liver Homogenate Preparation:

The minced pieces of liver tissues were transferred to tared 50 mLcentrifuge tubes and weighed. Chilled homogenization buffer (100 mM TrispH 8.0, 1 mM magnesium acetate, with antibiotic-antimycotic agents) wasadded to each tube, such that the tube(s) contained 5 mL of buffer pergram of tissue. Using a QIAGEN TissueRuptor tissue homogenizer, theliver/buffer mixture was homogenized while maintaining the tube on ice.The protein concentration of the liver homogenate pool was determinedusing a Pierce BCA protein assay. Liver homogenates were divided into 5mL aliquots, transferred to appropriately sized labeled cryovials andstored at −60° C.

Incubation Conditions:

5 ml aliquots of frozen liver homogenate (protein concentration=22.48mg/ml) were thawed and incubated at 37° C. for 24 hrs. Six eppendorftubes (2 ml) were taken for each oligomer in table land 450 ul ofhomogenate was added in each tube. 50 ul ASO (200 uM) was added to eachtube. Immediately after mixing, 125 ul of (5×) stop buffer (2.5% IGEPAL,0.5M NaCl, 5 mM EDTA, 50 mM Tris, pH=8.0) and 12.5 ul of 20 mg/mlProteinase K (Ambion, # AM2546) was added to one tube for 0 hour timepoint. The remaining reaction mixtures were incubated at 37° C. withshaking at 400 rpm on VWR Incubating Microplate shaker. After incubationfor a designated period (1, 2, 3, 4, and 5 days), each mixture wastreated with 125 ul of (5×) stop buffer (2.5% IGEPAL, 0.5M NaCl, 5 mMEDTA, 50 mM Tris, pH=8.0) and 12.5 ul of 20 mg/ml Proteinase K (Ambion,# AM2546).

Work up and Bioanalysis: ISIS 355868 (5′-GCGTTT√{square root over(GCTCTT)}CTTCTTGCGTTTTTT-3′ (SEQ ID NO: 392)), a 27-mer oligonucleotide(underlined bases are MOE modified) was used as the internal standardfor quantitation of chiromersens. 50 ul of internal standard (200 uM)was added to each tube followed by addition of 250 ul of 30% ammoniumhydroxide, 800 ul of Phenol: Chloroform: isoamyl alcohol (25:24:1).After mixing and centrifugation at 600 rpg, the aqueous layer wasevaporated on speed vac to 100 ul and loaded on Sep Pak column (C18, 1g, WAT 036905). All the aqueous washings (2×20 ml) of Sep pak columnwere tested with quick Ion Exchange method to ensure that no product wasfound there. 50% ACN (3.5 ml) was used to elute the oligonucleotide andmetabolites and the column was further washed with 70% CAN (3.5) ensurethat there was nothing left on the column. Five fractions were collectedfor each sequence. Water wash 1, 2, 3, ACN1 and 2 using Visiprep system(Sigma, part number: 57031-U).

Ion Exchange Method

Time Flow (ml/min) % A % B Curve Time 1.0 95 5 1 2 1.0 95 5 1 2 3 1.0 7525 6 3 10 1.0 35 65 6 4 10.1 1.0 95 5 6 5 12.5 1.0 95 5 1Buffer A=10 mM Tris HCl, 50% ACN, pH=8.0

Buffer B=A+800 mM NaClO4 Column=DNA pac 100 Column Temperature 60° C.

Wash method was used after each run (Described in M9-Exp21) using thesame buffers as above and 50:50 (methanol:water) in buffer line C.

Time Flow (ml/min) % A % B % C Curve Time 1.0 0 0 100 1 5.5 1.0 0 0 1001 2 5.6 1.0 100 0 0 6 3 7.5 1.0 100 0 0 6 4 7.6 1.0 95 5 0 6 5 12.5 1.095 5 0 1Acetonitrile eluate was concentrated to dryness and dissolved in 100 ulwater to be analyzed using RPHPIPC.Eluant A=10 mM Tributylammonium acetate, pH=7.0Eluant B=ACN (HPLC grade, B& J)Column: XTerra MS C18, 3.5 um, 4.6×50 mm, Part number: 186000432Guard column from Phenomenex, part number: KJ0-4282 ColumnTemperature=60° C.

HPLC Gradient:

Time Flow (ml/min) % A % B Curve 1 1.0 65 35 2 5.0 1.0 65 35 1 3 30.01.0 40 60 6 4 35.0 1.0 5 90 6 5 36.0 1.0 65 35 6 6 40.0 1.0 65 35 1For Analytical RP HPLC, 10 ul of this stock solution was added to 40 ulwater and 40 ul was injected.

TABLE 3 S.NO. Sequence Description ONT- Gs5mCs5mCs Ts5mCsAs GsTs5mCsTsGs5mCs TsTs5mCs Gs5mCsAs 5mCs5mC Mipomersen 41 (SEQ ID NO: 393) ONT-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC MOE-wing- 87(SEQ ID NO: 394) core-wing design - (human) RNAse H substrate 15R-(SSR)₃-5R ONT-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC All deoxy,(5S- 154 (SEQ ID NO: 395) (SSR)₃-5S) ONT-Gs5mCsGsTsTsTsGs5mCsTs5mCsTsTs5mCsTsTs5mCsTsTsGs5mCGsTsTsTsTsTsT ISIS355868 70 (SEQ ID NO: 396) internal standard for quantitation ofMipomersen

Discussion:

2′ modifications in antisense and siRNAs are predicted to stabilizethese molecules and increase their the persistence in plasma and tissuescompared with wild-type DNAs and siRNAs.

2′-MOE Wing-Core-Wing Design in Mipomersen.

The first generation antisense oligonucleotides employed in the firstantisense clinical trials had 2′-deoxy ribonucleotide residues andphosphorothioate internucleoside linkages. Subsequently, secondgeneration antisense oligonucleotides were developed, which weretypically of what is referred to herein as “5-10-5 2′-MOE wing-core-wingdesign”, in that five (5) residues at each end were 2′-O-methoxyethyl(2′-MOE)-modified residues and ten (10) residues in the middle were2′-deoxy ribonucleotides; the internucleotide linkages of sucholigonucleotides were phosphorothioates. Such “5-10-5 2′-MOEwing-core-wing” oligonucleotides exhibited marked improvement in potencyover first generation (PCT/US2005/033837). Similar wing-core-wing motifslike 2-16-2, 3-14-3, 4-12-4, or 5-10-5 were designed to improve thestability of oligonucleotides to nucleases, while at the same timemaintaining enough DNA structure for RNase activity.

Chirally pure oligonucleotides. The present disclosure provides chirallypure oligonucleotides and demonstrates, among other things, thatselection of stereochemistry in and of itself can improveoligonucleotide stability (i.e., independent of residue modificationsuch as 2′MOE modification). Indeed, the present disclosure demonstratesthat chirally pure phosphorothioate oligonucleotides can provide same orbetter stability than corresponding 2′-modified stereorandomphosphorothioate compounds.

In some embodiments, tested chirally pure oligonucleotides are of thegeneral structure X—Y—X with respect to stereochemistry in that theycontain wing “X” regions (typically about 1-10 residues long) where allresidues have the same stereochemistry flanking a core “Y” region inwhich stereochemistry varies. In many embodiments, about 20-50% of thenucleotide analogs in tested such oligonucleotides are not substratesfor RNase H. The ability to control the stereochemistry ofphosphorothioates in DNA enables us to protect the oligomers fromdegradation by nucleases while maintaining the RNase active sites. Oneof these designs is ONT-154 where wings of the oligonucleotide have beenstabilized by Sp phosphorothioate chemistry with retention of few Rpphosphorothioates which are better substrates for RNase H (MolecularCell, 2007). The crystal structure of human RNase H complexed withDNA/RNA duplex shows that the Phosphate-binding pocket of the enzymemakes contacts with four contiguous phosphates of DNA. The first threecontacts seem stronger than fourth one and they prefer Pro-R/Pro-R/Pro-Soxygen atoms of each of these three phosphates. Combining the stabilityadvantage coming from Sp stereochemistry with RNase H active sites,several sequences can be designed to compete with/or improve upon2′-modifications. From rat whole liver homogenate stability experimentcomparing Mipomersen (ONT-41) with our rational (chiral control) designwith and without 2′-modifications (ONT-87 and ONT-154) (Table 1 and FIG.1), it is evident that through removal of the 2′-modifications andcareful chiral control with Rp and Sp phosphorothioates, we can improvethe stability of these oligonucleotides which later affect the efficacyin vivo.

TABLE 4 Hu chiromersens studied for rat whole live homogenate stabilitySEQ ID Sequence Description Target Tm (° C.) NO: ONT-41Gs5mCs5mCs Ts5mCsAs GsTs5mCs TsGs5mCs Hu ApoB 80.7 397 TsTs5mCsGs5mCsAs 5mCs5mC ONT-75 Gs5mCs5mCsTs5mC sAsGsTs5mCsTsGs5mCsTs Hu ApoB85.0 398 Ts5mCs Gs5mCsAs5mCs5mC ONT-77 Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTs Hu ApoB 79.9 399 Ts5mCs Gs5mCsAs5mCs5mC ONT-80Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsT Hu ApoB 75.8 400 s5mCsGs5mCsAs5mCs5mC ONT-81 Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsT Hu ApoB80.7 401 s5mCs Gs5mCsAs5mCs5mC ONT-87Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB 82.4 402 s5mCsGs5mCsAs5mCs5mC ONT-88 Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB78.9 403 s5mCs Gs5mCsAs5mCs5mC ONT-89Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsT Hu ApoB 80.9 404 s5mCsGs5mCsAs5mCs5mC ONT-70 Gs5mCsGsTsTsTsGs5mCsTs5mCsTsTs5mCsTsTs ISIS 4055mCsTsTsGs5mCGsTsTsTsTsTsT 355868 internal standard

TABLE 5 Mouse chiromersens studied for rat whole live homogenatestability SEQ ID Sequence Description Target NO: ONT-83 GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 406 ApoB ONT-82GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 407 ApoBONT-84 GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse 408ApoB ONT-85 GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC Mouse409 ApoB ONT-86 GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mCMouse 410 ApoB

Example 2. Example Chirally Controlled siRNA Molecules

TABLE 1 Summary of Phosphodiester Polar interactions with h-Ago-2 andh-Ago-1 Science 2012 hAgo-2 Phosphate* Residue Length/Å Config 2 Asn5512.7 Pro(S) Gln548 2.9 Pro(S) 3 Lys566 3.1 Pro(R) Arg792 3.4 Pro(R) 4Tyr790 2.6 Pro(R) Arg792 3.0 Pro(R) 2.8 Pro(R) 3.4 Pro(S) 5 Ser798 2.7Pro(R) 2.9 Pro(R) Tyr804 2.8 Pro(S) 6 Lys709 3.0 Pro(S) Arg761 2.9Pro(R) His753 2.8 Pro(R) 7 Arg714 2.9 Pro(R) 3.0 Pro(R) Arg761 3.0Pro(S) *Phosphate No. from 5′-end Cell 2012 hAgo-2 Phosphate ResidueLength/Å Config 2 Asn551 2.7 Pro(S) Gln548 3.1 Pro(S) Gln548 2.9 Pro(R)3 Lys566 2.9 Pro(R) Arg792 3.3 Pro(R) 4 Tyr790 2.8 Pro(R) Arg792 2.8Pro(R) 5 Ser798 2.6 Pro(R) 2.9 Pro(R) Tyr804 2.5 Pro(S) 6 Lys709 3.2Pro(S) Arg761 2.8 Pro(R) His753 3.0 Pro(R) 7 Arg714 2.8 Pro(R) 3.1Pro(R) Arg761 2.8 Pro(S) 8 Arg761 2.4 Pro(S) Ala221 3.5 Pro(R) 9 Arg3512.2 Pro(R) 10 Arg710 2.5 Pro(R) 18 No contacts 19 Tyr311 3.1 Pro(R)Arg315 2.8 Pro(R) 20 His271 3.1 Pro(R) His319 3.4 Pro(S) Tyr311 2.2Pro(S) Cell Rep 2013, h-Ago-1^(†) Phosphate Residue Length/Å Config 2Asn549 2.7 Pro(S) Gln546 2.9 Pro(S) 2.8 Pro(R) 2 Lys564 2.9 Pro(R)Arg790 3.4 Pro(R) 3.3 Pro(R) 4 Try788 2.7 Pro(R) Arg790 3.3 Pro(R) 5Ser796 2.5 Pro(R) 2.8 Pro(R) Tyr802 2.6 Pro(S) 6 Lys707 2.8 Pro(S)Arg759 2.7 Pro(R) His751 3.0 Pro(R) 7 Arg712 3.1 Pro(S) 3.3 Pro(S)Arg373 3.4 Pro(R) Thr757 2.9 Pro(R) 8 Arg759 2.2 Pro(S) His710 3.4Pro(R) Ser218 2.7 Pro(R) 9 Arg349 3.5 Pro(R) Arg708 2.9 Pro(S) 10 Arg7083.2 Pro(R) 2.9 Pro(R) 21 Tyr309 2.6 Pro(S) Tyr314 2.6 Pro(S) His269 3.0Pro(R) ^(†)Complexed with h-let-7 22mer

The present disclosure, despite teachings in the art to the contrary,recognizes that stereochemistry of internucleotidic linkages can beutilized to increase stability and activity of oligonucleotides throughchirally controlled oligonucleotide compositions. Such chirallycontrolled oligonucleotide compositions can provide much better resultsthan chirally uncontrolled oligonucleotide compositions as demonstratedin this disclosure.

There are two reported crystal structures of RNA complexed with humanArgonaute-2 protein (hAgo2): The Crystal Structure of Human Argonaute-2,Science, 2012 (PDB-4ei3); and The Structure of Human Argonaute-2 inComplex with miR-20a Cell, 2012 PDB-4f3t). In addition, there is onereported crystal structure of Let-7 RNA complexed with human Argonaute-1protein (hAgo-1): The Making of a Slicer: Activation of HumanArgonaute-1, Cell Rep. 2013 (PDB-4krf).

Based upon the information contained in these publications, it wasanticipated that some judgments could be made about advantageouspreferences for stereochemistry at the internucleotidic phosphatelinkage if the phosphodiester bonds were to be replaced byphosphorothioate diester bonds. These advantages could relate tosignificantly improved potency, stability and other pharmacologicalproperties. With this in mind, the computer program Pymol was used tolocate all polar interactions between the protein and theinternucleotidic phosphodiester linkage of the crystallized RNA for allthree structures. Polar interactions at a distance of more than 3.5 Awere ignored.

The results of this analysis are summarized in Table 1. A particularphosphorus atom from the phosphodiester backbone on the RNA was assigneda Pro(R) or a Pro(S) configuration based upon the assumption that in thephosphorothioate diester analog the quite similar bond would be madebetween the polar group on an amino acid residue and the respectfulphosphate oxygen atom. The sulfur substitution, instead of non-bridgingoxygen would therefore confer a unique stereochemistry (either (Sp) or(Rp) absolute configuration) on the phosphorus atom within that motif.

Of note is the extraordinarily good agreement between the two structuresof hAgo-2 in complex with RNA. Also, there is an excellent agreementbetween the structures of hAgo-1 and hAgo-2 in complex with RNA,indicating that the conformation that the RNA molecule adopts is highlyconserved between these two proteins. Any conclusions or rules which areformed based upon the results of this analysis are likely, therefore, tobe valid for both protein molecules.

As can be seen, there is usually more than one polar interaction at anyone phosphodiester group, with the exception of those between thephosphodiesters at phosphate positions 9 and 10 and hAgo-2 (Cell 2012)which adopt exclusively Pro(Rp) preference through bonding with Arg351and Arg710 respectively.

However, shorter distances (corresponding to stronger interactions) aswell as the number of bonds per oxygen can suggest a predominantinteraction for the Pro(Rp) or the Pro(Sp) oxygens: hence resulting inseveral interactions which are predominantly of one stereochemical typeor the other. Within this group are the interactions between thephosphodiesters at phosphate positions 2 (Sp), 3 (Rp), 4 (Rp), 6 (Rp), 8(Sp), 19 (Rp), 20 (Sp) and 21 (Sp).

Of the remaining interactions, there does not appear to be a preferencefor one particular stereochemistry to be adopted over the other, so thepreferred stereochemistry could be either (Sp) or (Rp).

Within this category are the interactions formed between thephosphodiesters at phosphate positions 5 (Rp or Sp) and 7 (Rp or Sp).

For interactions at the other phosphate backbone, there is no crystalstructure information, so stereochemistry at these positions cansimilarly be either (Rp) or (Sp) until empirical data shows otherwise.

To this end, Table 6 contains several non-limiting example siRNA generalconstructs which can be conceived to take advantage of this preferencefor stereochemistry at individual phosphorothioate diester motifs.

TABLE 6 Example general siRNA constructs PS* Chirally ControlledAntisense Strand Construct 2 (Sp) (Rp) (Sp) (Rp) (Sp) (Rp) 3 (Rp) (Sp)(Rp) (Sp) (Rp) (Sp) 4 (Rp) (Sp) PO PO (Rp) (Sp) 5 (Rp) or (Sp) (Sp) or(Rp) PO PO PO PO 6 (Rp) (Sp) PO PO (Rp) (Sp) 7 (Rp) or (Sp) (Sp) or (Rp)PO PO PO PO 8 (Sp) (Rp) PO PO (Sp) (Rp) 9 (Rp) (Sp) PO PO (Rp) (Sp) 10(Rp) (Sp) PO PO (Rp) (Sp) 11 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 12(Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 13 (Rp) or (Sp) (Sp) or (Rp) PO POPO PO 14 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 15 (Rp) or (Sp) (Sp) or(Rp) PO PO PO PO 16 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO 17 (Rp) or(Sp) (Sp) or (Rp) PO PO PO PO 18 (Rp) or (Sp) (Sp) or (Rp) PO PO PO PO19 (Rp) (Sp) PO PO (Rp) (Sp) 20 (Sp) (Rp) (Sp) (Rp) (Sp) (Rp) 21 (Sp)(Rp) (Sp) (Rp) (Sp) (Rp) *The number indicates the phosphate positionfrom the 5′ end of the antisense strand of the siRNA, (e.g. #2 islocated between nucleotides 1 and 2 and #21 is located betweennucleotides 20 and 21). (Sp) and (Rp) designates stereochemistry ofphosphorus atom on phosphorothioate (PS) diester internucleotidiclinkage at the indicated position. PO designates a phosphodiesterinternucleotidic linkage at the indicated position.

Example siRNAs include but are not limited to siRNAs having a Spconfiguration for a chiral phosphorothioate at the 3′end and at the5′end of the antisense strand of the siRNA duplex, which confersunprecedentedly increased stability in human serum or biological fluids.That same Sp configuration for the chiral phosphorothioate at the 3′endand at the 5′end of the antisense strand of the siRNA duplex confersunprecedentedly increased biological potency caused by increasedaffinity to the Ago2 protein leading to increased activity within theRISC RNAi silencing complex.

In one embodiment, a single chiral phosphorothioate motif is introducedindependently at each position along the antisense or sense strand ofthe siRNA molecule. For a 21mer, this provides 80 unique sequences, witheither an (Sp) or an (Rp) chirally controlled phosphorothioate group.When duplexed independently, 1600 unique combinations of siRNAs areprepared.

siRNA Transfection of Chiral siRNA Molecules

Hep3B, or HeLa cells are reverse transfected at a density of 2.0×10⁴cells/well in 96-well plates. Transfection of siRNA is carried out withlipofectamine RNAiMax (Life Technologies, cat. No. 13778-150) using themanufacturer's protocol, except with a decreased amount of LipofectamineRNAiMax of 0.2 ul per well. Twelve, 1:3 siRNA duplex dilutions arecreated starting at 1 uM. 10 ul of 10×siRNA duplex is then lipoplexedwith a prepared mixture of 9.8 ul of serum-free medium and 0.2 ul ofLipofectamine RNAiMax per well. After a 10-15 minute incubation, 2.0×10⁴cells in 80 ul of EMEM cell growing media (ATCC, 30-2003) is added tobring the final volume to 100 ul per well. Two separate transfectionevents are performed for each dose.

24 hours after transfection Hep3B or HeLa cells are lysed and mRNAagainst which the siRNA is targeted is purified using MagMAX™-96 TotalRNA Isolation Kit (Life Technologies, AM1830); 15 ul of cDNA issynthesized with High Capacity cDNA Reverse Transcription Kit with RNaseInhibitor (Life Technologies, 4374967). Gene expression is evaluated byReal-Time PCR on a Lightcycler 480(Roche) using a Probes Master Mix(Roche, 04 707 494 001) according to manufacturer's protocol.

IC50s and Data Analysis

Delta Ct method is used to calculate values. Samples are normalized tohGAPDH and calibrated to mock transfected and untreated samples. Astereo-random molecule is used as a control. The data is represented asa mean of 2 biological replicates using Graphpad Prism. A four-parameterlinear regression curve is fitted to the data and the bottom and top areconstrained to a 0 and 100 constants respectively in order to calculatea relative IC50.

The present Example demonstrates successful inhibition of target geneexpression using siRNA agents comprised of chirally controlledoligonucleotides as described herein. Specifically, this Exampledescribes hybridization of individual oligonucleotide strands preparedthrough chirally controlled synthesis as described herein, so thatdouble-stranded chirally controlled siRNA oligonucleotide compositionsare provided. This Example further demonstrates successful transfectionof cells with such agents and, moreover, successful inhibition of targetgene expression.

In Vitro Metabolic Stabilities of Human PCSK9 siRNA Duplexes HavingStereocontrolled Phosphorothioate Diester Linkages in Human Serum.

10 μM siRNA duplexes were incubated in 90% human serum (50 μL, Sigma,H4522) at 37° C. for 24 hours. A 0 min time point (50 μL) was preparedas well as a PBS control incubation time point (50 μL), where the 10 μMsiRNA duplex was incubated in 90% 1×PBS (50 μL at 37° C. for 24 hours.After completion of the incubation, to each time point, were added 10 μLof Stop-Solution (0.5 M NaCl, 50 mM TRIS, 5 mM EDTA, 2.5% IGEPAL),followed by 3.2 μL of Proteinase K (20 mg/mL, Ambion). The samples wereincubated at 60° C. for 20 min, and then centrifuged at 2000 rpm for 15min. The final reaction mixtures were directly analyzed in denaturingIEX HPLC (injection volume 50 μL). The ratio of integrated area at 24 hand 0 min was used to determine the % of degradation for each siRNA.

It was observed that the stereochemistry configuration of the singlephosphorothioate at position 21 (3′end) of both the antisense strand andthe sense strand of the siRNA had a crucial impact on the stability ofthe duplex upon incubation in Human Serum (FIG. 1). As illustrated inthe FIG. 1 and as determined following the integration ratio of thedegradation pattern, an (Rp, Rp) siRNA duplex exhibited a significant55.0% degradation after 24 h. The stereorandom mixture ofphosphorothioates in the stereorandom siRNA showed 25.2% degradationafter 24 h. The (Sp/Sp) siRNA showed only minor 7.3% degradation after24 h. This illustrates the drastic impact that phosphorothioatestereochemistry confers to therapeutic siRNAs. Additional example datawere presented in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.

It is observed that each of the stereopure constructs show differentpotency (IC₅₀ values) dependent on the position of the phosphorothioatemotif along the backbone. It is also observered that different IC₅₀values are obtained dependent upon whether the phosphorothioate motif atany single position is (Sp) or (Rp). The impact of stereochemistry uponstability is likewise clear and differentiating, using either HumanSerum described above, or Human Hepatic Cytosol extract or Snake VenomPhosphodiesterase, or isolated endonuclease or isolated exonuclease.

Certain design rules may be formulated based upon data obtained in theabove example. These design information can be applied for theintroduction of multiple chiral phosphorothioate linkages within theantisense and/or sense strand of the siRNA as exemplified below. Thepresent disclosure recognizes that an increased amount of chiralphosphorothioate within the antisense and/or sense strand of the siRNA,introduced at the right positions and having the right stereochemistryconfiguration leads to greatly improved siRNA constructs in terms ofpotency and metabolic stability in vitro—translating into greatlypharmacologically enhanced therapeutic siRNAs.

Example Chirally Controlled siRNA Oligonucleotides Targeting PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9), is an enzymeinvolved in cholesterol metabolism. PCSK9 binds to the receptor for lowdensity lipoprotein (LDL), triggering its destruction. Although LDLassociated with the receptor is also eliminated when the receptor isdestroyed, the net effect of PCSK9 binding in fact increases LDL levels,as the receptor would otherwise cycle back to the cell surface andremove more cholesterol.

Several companies are developing therapeutic agents that target PCSK9.Of particular relevance to the present disclosure, each of IsisPharmaceuticals, Santaris Pharma, and Alnylam Pharmaceuticals isdeveloping a nucleic acid agent that inhibits PCSK9. The IsisPharmaceuticals product, an antisense oligonucleotide, has been shown toincrease expression of the LDLR and decrease circulating totalcholesterol levels in mice (Graham et al “Antisense inhibition ofproprotein convertase subtilisin/kexin type 9 reduces serum LDL inhyperlipidemic mice”. J. Lipid Res. 48 (4): 763-7, April 2007). Initialclinical trials with the Alnylam Pharmaceuticals product, ALN-PCS,reveal that RNA interference offers an effective mechanism forinhibiting PCSK9 (Frank-Kamenetsky et al “Therapeutic RNAi targetingPCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterolin nonhuman primates”. Proc. Natl. Acad. Sci. U.S.A. 105 (33): 11915-20,August 2008).

In some embodiments, despite known results to the contrary, the presentdisclosure recognizes that phosphorothioate motifs of one stereochemicalconformation or another can be rationally designed to take advantage ofincreased potency, stability and other pharmacological qualities throughchirally controlled oligonucleotide compositions. To reinforce thisconcept, table 3 contains example stereochemically pure constructs basedon an siRNA sequence which targets PCSK9 messenger RNA.

In this example embodiment, a single chiral phosphorothioate motif isintroduced independently at each position along the antisense or sensestrand of the siRNA molecule. For a 21mer, this provides 80 uniquesequences, with either an (Sp) or an (Rp) chirally controlledphosphorothioate group. When duplexed independently, 1600 uniquecombinations of siRNAs are prepared.

In other example embodiments, a single chiral phosphorothioate motif isintroduced independently at each position along the antisense or sensestrand of the siRNA molecule, while a 3′-(Sp) phosphorothioate linkageis conserved. For a 21mer, this provides another additional 80 uniquesequences, with either an (Sp) or an (Rp) chirally controlledphosphorothioate group. When duplexed independently, 1600 uniquecombinations of siRNAs are prepared.

In other example embodiments, multiple chiral phosphorothioate motifsare introduced independently at several positions along the antisense orsense strand of the siRNA molecule, following the codes described inTable 7, while a 3′-(Sp) phosphorothioate linkage is conserved.

TABLE 7 Example of PCSK-9 Sense and Antisense RNAs SEQ PCSK9 siRNA SenseStrands ID NO: PCSK9 (1) (Rp)-uucuAGAccuGuuuuGcuudTsdT 411 PCSK9 (2)(Sp)-uucuAGAccuGuuuuGcuudTsdT 412 PCSK9 (3)(Rp)-uucuAGAccuGuuuuGcuusdTdT 413 PCSK9 (4)(Sp)-uucuAGAccuGuuuuGcuusdTdT 414 PCSK9 (5)(Rp)-uucuAGAccuGuuuuGcusudTdT 415 PCSK9 (6)(Sp)-uucuAGAccuGuuuuGcusudTdT 416 PCSK9 (7)(Rp)-uucuAGAccuGuuuuGcsuudTdT 417 PCSK9 (8)(Sp)-uucuAGAccuGuuuuGcsuudTdT 418 PCSK9 (9)(Rp)-uucuAGAccuGuuuuGscuudTdT 419 PCSK9 (10)(Sp)-uucuAGAccuGuuuuGscuudTdT 420 PCSK9 (11)(Rp)-uucuAGAccuGuuuusGcuudTdT 421 PCSK9 (12)(Sp)-uucuAGAccuGuuuusGcuudTdT 422 PCSK9 (13)(Rp)-uucuAGAccuGuuusuGcuudTdT 423 PCSK9 (14)(Sp)-uucuAGAccuGuuusuGcuudTdT 424 PCSK9 (15)(Rp)-uucuAGAccuGuusuuGcuudTdT 425 PCSK9 (16)(Sp)-uucuAGAccuGuusuuGcuudTdT 426 PCSK9 (17)(Rp)-uucuAGAccuGusuuuGcuudTdT 427 PCSK9 (18)(Sp)-uucuAGAccuGusuuuGcuudTdT 428 PCSK9 (19)(Rp)-uucuAGAccuGsuuuuGcuudTdT 429 PCSK9 (20)(Sp)-uucuAGAccuGsuuuuGcuudTdT 430 PCSK9 (21)(Rp)-uucuAGAccusGuuuuGcuudTdT 431 PCSK9 (22)(Sp)-uucuAGAccusGuuuuGcuudTdT 432 PCSK9 (23)(Rp)-uucuAGAccsuGuuuuGcuudTdT 433 PCSK9 (24)(Sp)-uucuAGAccsuGuuuuGcuudTdT 434 PCSK9 (25)(Rp)-uucuAGAcscuGuuuuGcuudTdT 435 PCSK9 (26)(Sp)-uucuAGAcscuGuuuuGcuudTdT 436 PCSK9 (27)(Rp)-uucuAGAsccuGuuuuGcuudTdT 437 PCSK9 (28)(Sp)-uucuAGAsccuGuuuuGcuudTdT 438 PCSK9 (29)(Rp)-uucuAGsAccuGuuuuGcuudTdT 439 PCSK9 (30)(Sp)-uucuAGsAccuGuuuuGcuudTdT 440 PCSK9 (31)(Rp)-uucuAsGAccuGuuuuGcuudTdT 441 PCSK9 (32)(Sp)-uucuAsGAccuGuuuuGcuudTdT 442 PCSK9 (33)(Rp)-uucusAGAccuGuuuuGcuudTdT 443 PCSK9 (34)(Sp)-uucusAGAccuGuuuuGcuudTdT 444 PCSK9 (35)(Rp)-uucsuAGAccuGuuuuGcuudTdT 445 PCSK9 (36)(Sp)-uucsuAGAccuGuuuuGcuudTdT 446 PCSK9 (37)(Rp)-uuscuAGAccuGuuuuGcuudTdT 447 PCSK9 (38)(Sp)-uuscuAGAccuGuuuuGcuudTdT 448 PCSK9 (38)(Rp)-usucuAGAccuGuuuuGcuudTdT 449 PCSK9 (40)(Sp)-usucuAGAccuGuuuuGcuudTdT 450 NOTE: lower case letters represent2′-OMe RNA residues; capital letters represent RNA residues; d= 2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

Synthesis examples for Human PCSK9 siRNA Antisense Strands havingseveral chiral phosphorothioate internucleotide linkages and full chiralphosphorothioate internucleotide linkages.

Human PCSK9 siRNA Antisense Strands SEQ ID NO: PCSK9 (41)(Rp)-AAGcAAAAcAGGUCuAGAAdTsdT 451 PCSK9 (42)(Sp)-AAGcAAAAcAGGUCuAGAAdTsdT 452 PCSK9 (43)(Rp)-AAGcAAAAcAGGUCuAGAAsdTdT 453 PCSK9 (44)(Sp)-AAGcAAAAcAGGUCuAGAAsdTdT 454 PCSK9 (45)(Rp)-AAGcAAAAcAGGUCuAGAsAdTdT 455 PCSK9 (46)(Sp)-AAGcAAAAcAGGUCuAGAsAdTdT 456 PCSK9 (47)(Rp)-AAGcAAAAcAGGUCuAGsAAdTdT 457 PCSK9 (48)(Sp)-AAGcAAAAcAGGUCuAGsAAdTdT 458 PCSK9 (49)(Rp)-AAGcAAAAcAGGUCuAsGAAdTdT 459 PCSK9 (50)(Sp)-AAGcAAAAcAGGUCuAsGAAdTdT 460 PCSK9 (51)(Rp)-AAGcAAAAcAGGUCusAGAAdTdT 461 PCSK9 (52)(Sp)-AAGcAAAAcAGGUCusAGAAdTdT 462 PCSK9 (53)(Rp)-AAGcAAAAcAGGUCsuAGAAdTdT 463 PCSK9 (54)(Sp)-AAGcAAAAcAGGUCsuAGAAdTdT 464 PCSK9 (55)(Rp)-AAGcAAAAcAGGUsCuAGAAdTdT 465 PCSK9 (56)(Sp)-AAGcAAAAcAGGUsCuAGAAdTdT 466 PCSK9 (57)(Rp)-AAGcAAAAcAGGsUCuAGAAdTdT 467 PCSK9 (58)(Sp)-AAGcAAAAcAGGsUCuAGAAdTdT 468 PCSK9 (59)(Rp)-AAGcAAAAcAGsGUCuAGAAdTdT 469 PCSK9 (60)(Sp)-AAGcAAAAcAGsGUCuAGAAdTdT 470 PCSK9 (61)(Rp)-AAGcAAAAcAsGGUCuAGAAdTdT 471 PCSK9 (62)(Sp)-AAGcAAAAcAsGGUCuAGAAdTdT 472 PCSK9 (63)(Rp)-AAGcAAAAcsAGGUCuAGAAdTdT 473 PCSK9 (64)(Sp)-AAGcAAAAcsAGGUCuAGAAdTdT 474 PCSK9 (65)(Rp)-AAGcAAAAscAGGUCuAGAAdTdT 475 PCSK9 (66)(Sp)-AAGcAAAAscAGGUCuAGAAdTdT 476 PCSK9 (67)(Rp)-AAGcAAAsAcAGGUCuAGAAdTdT 477 PCSK9 (68)(Sp)-AAGcAAAsAcAGGUCuAGAAdTdT 478 PCSK9 (69)(Rp)-AAGcAAsAAcAGGUCuAGAAdTdT 479 PCSK9 (70)(Sp)-AAGcAAsAAcAGGUCuAGAAdTdT 480 PCSK9 (71)(Rp)-AAGcAsAAAcAGGUCuAGAAdTdT 481 PCSK9 (72)(Sp)-AAGcAsAAAcAGGUCuAGAAdTdT 482 PCSK9 (73)(Rp)-AAGcsAAAAcAGGUCuAGAAdTdT 483 PCSK9 (74)(Sp)-AAGcsAAAAcAGGUCuAGAAdTdT 484 PCSK9 (75)(Rp)-AAGscAAAAcAGGUCuAGAAdTdT 485 PCSK9 (76)(Sp)-AAGscAAAAcAGGUCuAGAAdTdT 486 PCSK9 (77)(Rp)-AAsGcAAAAcAGGUCuAGAAdTdT 487 PCSK9 (78)(Sp)-AAsGcAAAAcAGGUCuAGAAdTdT 488 PCSK9 (77)(Rp)-AsAGcAAAAcAGGUCuAGAAdTdT 489 PCSK9 (78)(Sp)-AsAGcAAAAcAGGUCuAGAAdTdT 490 PCSK9 (79) (Rp,Sp)-AAGcAAAAcAGGUCuAGAAsdTsdT 491 PCSK9 (80) (Sp,Sp)-AAGcAAAAcAGGUCuAGAAsdTsdT 492 PCSK9 (81) (Rp,Sp)-AAGcAAAAcAGGUCuAGAsAdTsdT 493 PCSK9 (82) (Sp,Sp)-AAGcAAAAcAGGUCuAGAsAdTsdT 494 PCSK9 (83) (Rp,Sp)-AAGcAAAAcAGGUCuAGsAAdTsdT 495 PCSK9 (84) (Sp,Sp)-AAGcAAAAcAGGUCuAGsAAdTsdT 496 PCSK9 (85) (Rp,Sp)-AAGcAAAAcAGGUCuAsGAAdTsdT 497 PCSK9 (86) (Sp,Sp)-AAGcAAAAcAGGUCuAsGAAdTsdT 498 PCSK9 (87) (Rp,Sp)-AAGcAAAAcAGGUCusAGAAdTsdT 499 PCSK9 (88) (Sp,Sp)-AAGcAAAAcAGGUCusAGAAdTsdT 500 PCSK9 (89) (Rp,Sp)-AAGcAAAAcAGGUCsuAGAAdTsdT 501 PCSK9 (90) (Sp,Sp)-AAGcAAAAcAGGUCsuAGAAdTsdT 502 PCSK9 (91) (Rp,Sp)-AAGcAAAAcAGGUsCuAGAAdTsdT 503 PCSK9 (92) (Sp,Sp)-AAGcAAAAcAGGUsCuAGAAdTsdT 504 PCSK9 (93) (Rp,Sp)-AAGcAAAAcAGGsUCuAGAAdTsdT 505 PCSK9 (94) (Sp,Sp)-AAGcAAAAcAGGsUCuAGAAdTsdT 506 PCSK9 (95) (Rp,Sp)-AAGcAAAAcAGsGUCuAGAAdTsdT 507 PCSK9 (96) (Sp,Sp)-AAGcAAAAcAGsGUCuAGAAdTsdT 508 PCSK9 (97) (Rp,Sp)-AAGcAAAAcAsGGUCuAGAAdTsdT 509 PCSK9 (98) (Sp,Sp)-AAGcAAAAcAsGGUCuAGAAdTsdT 510 PCSK9 (99) (Rp,Sp)-AAGcAAAAcsAGGUCuAGAAdTsdT 511 PCSK9 (100) (Sp,Sp)-AAGcAAAAcsAGGUCuAGAAdTsdT 512 PCSK9 (101) (Rp,Sp)-AAGcAAAAscAGGUCuAGAAdTsdT 513 PCSK9 (102) (Sp,Sp)-AAGcAAAAscAGGUCuAGAAdTsdT 514 PCSK9 (103) (Rp,Sp)-AAGcAAAsAcAGGUCuAGAAdTsdT 515 PCSK9 (104) (Sp,Sp)-AAGcAAAsAcAGGUCuAGAAdTsdT 516 PCSK9 (105) (Rp,Sp)-AAGcAAsAAcAGGUCuAGAAdTsdT 517 PCSK9 (106) (Sp,Sp)-AAGcAAsAAcAGGUCuAGAAdTsdT 518 PCSK9 (107) (Rp,Sp)-AAGcAsAAAcAGGUCuAGAAdTsdT 519 PCSK9 (108) (Sp,Sp)-AAGcAsAAAcAGGUCuAGAAdTsdT 520 PCSK9 (109) (Rp,Sp)-AAGcsAAAAcAGGUCuAGAAdTsdT 521 PCSK9 (110) (Sp,Sp)-AAGcsAAAAcAGGUCuAGAAdTsdT 522 PCSK9 (111) (Rp,Sp)-AAGscAAAAcAGGUCuAGAAdTsdT 523 PCSK9 (112) (Sp,Sp)-AAGscAAAAcAGGUCuAGAAdTsdT 524 PCSK9 (113) (Rp,Sp)-AAsGcAAAAcAGGUCuAGAAdTsdT 525 PCSK9 (114) (Sp,Sp)-AAsGcAAAAcAGGUCuAGAAdTsdT 526 PCSK9 (115) (Rp,Sp)-AsAGcAAAAcAGGUCuAGAAdTsdT 527 PCSK9 (116) (Sp,Sp)-AsAGcAAAAcAGGUCuAGAAdTsdT 528 PCSK9 (117) (Rp, Sp, Sp, Sp, Rp, Sp,Sp, Sp, Rp, Rp)- 529 AsAsGscAsAAsAscsAGGUCuAGAsAsdTsdT PCSK9 (118) (Sp,Rp, Rp, Rp, Sp, Rp, Rp, Rp, Sp, Sp)- 530AsAsGscAsAAsAscsAGGUCuAGAsAsdTsdT NOTE: lower case letters represent2′-OMe RNA residues; capital letters represent RNA residues; d= 2′-deoxy residues; and “s” indicates a phosphorothioate moiety.

Example 3. Stereopure FOXO—1 Antisense Analogs Rational Design—ChirallyControlled Antisense Oligonucleotide Compositions

The unprecedented nuclease stability determined in vivo and in a wholerat liver homogenate model of the Sp-chiral phosphorothioateinternucleotide linkage is applied in the novel design of new types ofRNaseH substrate gapmers, whereby the external flanks are composed ofunmodified DNA and the internal gap core is modified with 2′ chemicalmodifications (2′OMe, 2′MOE, 2′LNA, 2′F, etc). Eventually this design isextended to fully unmodified DNA therapeutic oligonucleotides whereincareful chiral control of the phosphorothioate backbone confers thedesired pharmacological properties of the RNaseH therapeuticoligonucleotide.

The application of the triplet-phosphate repeating motif designed afterstudying the crystal structure of human RNaseH has been employed aswell. The crystal structure of RNaseH has been previously published(Structure of Human RNase H1 Complexed with an RNA/DNA Hybrid: Insightinto HIV Reverse Transcription, Nowotny et al., Molecular Cell, Volume28, Issue 2, 264-276, 2007, pdb file: 2 qkb). Among other things, thepresent disclosure recognizes the importance of internucleotidic linkagestereochemistry of oligonucleotides, for example, in settings herein.Upon performing in silico analysis upon this structure using the programPymol, Applicant found that the phosphate-binding pocket of Human RNaseH1 makes polar contacts with three contiguous phosphates of thecomplexed DNA, and interacts preferentially with thePro-R/Pro-R/Pro-S(or with the Pro-S/Pro-S/Pro-R) respective oxygen atomsof each of these three phosphates. Based on this observation we designedtwo chiral architectures with repeating (RRS) and (SSR) tripletphosphorothioates motifs as designed RNase H substrates. Applicant alsodesigned other internucleotidic linkage stereochemical patterns. Asdemonstrated by example results provide herein, provided chirallycontrolled oligonucleotide compositions of oligonucleotide types thatcomprises certain backbone internucleotidic linkage patterns (patternsbackbone chiral centers) provides significantly increased activityand/or kinetics. Among others, a sequence of 5′-RSS-3′ backbone chiralcenters is particularly useful and delivers unexpected results asdescribed in the present disclosure.

The combination of increased Sp chiral backbone (for enzymatic stabilityand other pharmacologically advantageous properties) and (RRS) or (SSR)repeating triplet chiral backbone motifs (for enhancing the property asRNase H substrate) are also utilized in the novel designs.; “S”represents Sp-phosphorothioate linkage and “R” representsRp-phosphorothioate linkage.

Another alternative design is based on the increased amount of Sp chiralphosphorothioate backbone in extended repeating motifs such as:(SSSR)_(n), SR(SSSR)_(n), SSR(SSSR)_(n), SSR(SSSR)_(n); (SSSSR)_(n),SR(SSSSR)_(n), SSR(SSSSR)_(n), SSR(SSSSR)_(n), SSSR(SSSSR)_(n);(SSSSSR)_(n); SR(SSSSSR)_(n), SSR(SSSSSR)_(n), SSR(SSSSSR)_(n),SSSR(SSSSSR)_(n), SSSSR(SSSSSR)_(n); etc., where n=0-50, depending onthe number of respective internucleotide linkages.; “S” representsSp-phosphorothioate linkage and “R” represents Rp-phosphorothioatelinkage. In some embodiments, n is 0. In some embodiments, R is 1-50. Insome embodiments, R is 1. In some embodiments, a common pattern ofbackbone chiral centers of a provided chirally controlledoligonucleotide composition comprises a motif described herein. In someembodiments, a motif is in the core region. In some embodiments, n is 0.In some embodiments, R is 1-50. In some embodiments, R is 1. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5.

Another alternative design is based on the “invert” architecture designof the stereo backbone (“stereo invert-mers”). These result frompositioning the stereochemistry of the chiral phosphorothioate in ainverting manner, exposing some Sp-rich motifs at the 5′ and 3′ endextremities of the oligonucleotide as well as the middle portion of theoligonucleotide and having the repeating stereochemistry motifspositioned in a invert image manner on both sides, such as:

SS(SSR)_(n)(SSS)(RSS)_(n)SS;SS(SSR)_(n)(SRS)(RSS)_(n)SS;SS(SSR)_(n)(SSR)(RSS)_(n)SS;SS(SSR)_(n)(RSS)(RSS)_(n)SS;SS(RSS)_(n)(SSS)(SSR)_(n)SS;SS(RSS)_(n)(SRS)(SSR)_(n)SS;SS(RSS)_(n)(SSR)(SSR)_(n)SS;SS(RSS)_(n)(RSS)(SSR)_(n)SS; etc.,where n=0-50, depending on the number of respective internucleotidelinkages. “S” represents Sp-phosphorothioate linkage and “R” representsRp-phosphorothioate linkage. In some embodiments, a common pattern ofbackbone chiral centers of a provided chirally controlledoligonucleotide composition comprises a motif described herein. In someembodiments, a motif is in the core region. In some embodiments, n is 0.In some embodiments, n is 1. In some embodiments, n is 1-50. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5.

Initial Screen Synthesis: Summary for Oligonucleotide Synthesis on aDNA/RNA Synthesizer MerMade-12 (2′-Deoxy and 2′-OMe Cycle)

delivery step reaction reagent volume (mL) wait time (sec) 1detritylation 3% TCA in DCM 4 × 1     N.A. 2 coupling 0.15M 2 × 0.5 mL60 + 60 (DNA), phosphoramidite 300 + 300 (2′- in ACN + 0.45M OMe RNA)ETT in ACN 3 capping 5% Pac₂O in 1  60 THF/2,6- lutidine + 16% NMI inTHF 4 oxidation 0.02 Iodine in 1 240 water/pyridine

Stereorandom PS Oligonucleotides Having DNA-2′-OMe-DNA (7-6-7) Design

ONT-141 (SEQ ID NO: 531) d(CsCsCsTsCsTsGs)gsaststsgsasd(GsCsAsTsCsCsA)ONT-142 (SEQ ID NO: 532) d(AsAsGsCsTsTsTs)gsgststsgsgsd(GsCsAsAsCsAsC)ONT-143 (SEQ ID NO: 533) d(AsGsTsCsAsCsTs)tsgsgsgsasgsd(CsTsTsCsTsCsC)ONT-144 (SEQ ID NO: 534) d(CsAsCsTsTsGsGs)gsasgscststsd(CsTsCsCsTsGsG)ONT-145 (SEQ ID NO: 535) d(AsTsAsGsCsCsAs)tstsgscsasgsd(CsTsGsCsTsCsA)ONT-146 (SEQ ID NO: 536) d(TsGsGsAsTsTsGs)asgscsastscsd(CsAsCsCsAsAsG)ONT-147 (SEQ ID NO: 537) d(CsCsAsTsAsGsCs)csaststsgscsd(AsGsCsTsGsCsT)ONT-148 (SEQ ID NO: 538) d(GsTsCsAsCsTsTs)gsgsgsasgscsd(TsTsCsTsCsCsT)ONT-149 (SEQ ID NO: 539) d(CsCsAsGsGsGsCs)ascstscsastsd(CsTsGsCsAsTsG)ONT-150 (SEQ ID NO: 540) d(GsCsCsAsTsCsCs)asasgstscsasd(CsTsTsGsGsGsA)ONT-151 (SEQ ID NO: 541) d(GsAsAsGsCsTsTs)tsgsgststsgsd(GsGsCsAsAsCsA)ONT-152 (SEQ ID NO: 542) d(CsTsGsGsAsTsTs)gsasgscsastsd(CsCsAsCsCsAsA)ONT-183 (SEQ ID NO: 543) d(CsAsAsGsTsCsAs)cststsgsgsgsd(AsGsCsTsTsCsT)ONT-184 (SEQ ID NO: 544) d(AsTsGsCsCsAsTs)cscsasasgstsd(CsAsCsTsTsGsG)ONT-185 (SEQ ID NO: 545) d(AsTsGsAsGsAsTs)gscscstsgsgsd(CsTsGsCsCsAsT)ONT-186 (SEQ ID NO: 546) d(TsTsGsGsGsAsGs)cststscstscsd(CsTsGsGsTsGsG)ONT-187 (SEQ ID NO: 547) d(TsGsGsGsAsGsCs)tstscstscscsd(TsGsGsTsGsGsA)ONT-188 (SEQ ID NO: 548) d(TsTsAsTsGsAsGs)astsgscscstsd(GsGsCsTsGsCsC)ONT-189 (SEQ ID NO: 549) d(GsTsTsAsTsGsAs)gsastsgscscsd(TsGsGsCsTsGsC)ONT-190 (SEQ ID NO: 550) d(CsCsAsAsGsTsCs)ascststsgsgsd(GsAsGsCsTsTsC)ONT-191 (SEQ ID NO: 551) d(AsGsCsTsTsTsGs)gststsgsgsgsd(CsAsAsCsAsCsA)ONT-192 (SEQ ID NO: 552) d(TsAsTsGsAsGsAs)tsgscscstsgsd(GsCsTsGsCsCsA)ONT-193 (SEQ ID NO: 553) d(TsGsTsTsAsTsGs)asgsastsgscsd(CsTsGsGsCsTsG)ONT-194 (SEQ ID NO: 554) d(AsTsCsCsAsAsGs)tscsascststsd(GsGsGsAsGsCsT)ONT-195 (SEQ ID NO: 555) d(GsGsGsAsAsGsCs)tststsgsgstsd(TsGsGsGsCsAsA)ONT-196 (SEQ ID NO: 556) d(CsTsCsCsAsTsCs)csastsgsasgsd(GsTsCsAsTsTsC)ONT-197 (SEQ ID NO: 557) d(AsAsGsTsCsAsCs)tstsgsgsgsasd(GsCsTsTsCsTsC)ONT-198 (SEQ ID NO: 558) d(CsCsAsTsCsCsAs)asgstscsascsd(TsTsGsGsGsAsG)ONT-199 (SEQ ID NO: 559) d(TsCsCsAsAsGsTs)csascststsgsd(GsGsAsGsCsTsT)ONT-200 (SEQ ID NO: 560) d(CsCsTsCsTsGsGs)aststsgsasgsd(CsAsTsCsCsAsC)ONT-201 (SEQ ID NO: 561) d(AsCsTsTsGsGsGs)asgscststscsd(TsCsCsTsGsGsT)ONT-202 (SEQ ID NO: 562) d(CsTsTsGsGsGsAs)gscststscstsd(CsCsTsGsGsTsG)ONT-203 (SEQ ID NO: 563) d(CsAsTsGsCsCsAs)tscscsasasgsd(TsCsAsCsTsTsG)ONT-204 (SEQ ID NO: 564) d(TsGsCsCsAsTsCs)csasasgstscsd(AsCsTsTsGsGsG)ONT-205 (SEQ ID NO: 565) d(TsCsCsAsTsCsCs)astsgsasgsgsd(TsCsAsTsTsCsC)ONT-206 (SEQ ID NO: 566) d(AsGsGsGsCsAsCs)tscsastscstsd(GsCsAsTsGsGsG)ONT-207 (SEQ ID NO: 567) d(CsCsAsGsTsTsCs)cststscsastsd(TsCsTsGsCsAsC)ONT-208 (SEQ ID NO: 568) d(CsAsTsAsGsCsCs)aststsgscsasd(GsCsTsGsCsTsC)ONT-209 (SEQ ID NO: 569) d(TsCsTsGsGsAsTs)tsgsasgscsasd(TsCsCsAsCsCsA)ONT-210 (SEQ ID NO: 570) d(GsGsAsTsTsGsAs)gscsastscscsd(AsCsCsAsAsGsA)

Biology In Vitro Data in HepG2 Cells for the Initial DNA-2′-OMe-DNA(7-6-7) Design: (d Upper Case)=DNA; Lower Case=2′-OMe;s=Phosphorothioate

FOXO1 Levels at 20 nM (%) SD ONT-141 89 6 ONT-142 45 1 ONT-143 98 2ONT-144 89 1 ONT-145 46 5 ONT-146 99 1 ONT-147 66 6 ONT-148 101 2ONT-149 95 6 ONT-150 58 4 ONT-151 41 5 ONT-152 84 5 ONT-183 95 2 ONT-18458 4 ONT-185 42 2 ONT-186 96 4 ONT-187 92 3 ONT-188 47 5 ONT-189 63 5ONT-190 83 2 ONT-191 58 4 ONT-192 46 2 ONT-193 58 2 ONT-194 76 1 ONT-19566 0 ONT-196 77 2 ONT-197 90 6 ONT-198 42 4 ONT-199 68 1 ONT-200 89 6ONT-201 91 2 ONT-202 94 2 ONT-203 86 1 ONT-204 58 2 ONT-205 75 3 ONT-20694 5 ONT-207 96 0 ONT-208 54 0 ONT-209 87 4 ONT-210 92 4 Levels at 200nM (%) SD ONT-141 37 4 ONT-142 45 4 ONT-143 46 2 ONT-144 42 5 ONT-145 534 ONT-146 31 2 ONT-147 28 8 ONT-148 45 4 ONT-149 29 5 ONT-150 32 6ONT-151 38 4 ONT-152 30 5 ONT-183 60 5 ONT-184 34 2 ONT-185 50 2 ONT-18686 3 ONT-187 76 6 ONT-188 50 5 ONT-189 38 2 ONT-190 51 1 ONT-191 43 5ONT-192 54 7 ONT-193 41 6 ONT-194 50 1 ONT-195 43 6 ONT-196 33 7 ONT-19757 4 ONT-198 40 5 ONT-199 50 5 ONT-200 28 9 ONT-201 46 6 ONT-202 57 9ONT-203 27 7 ONT-204 36 6 ONT-205 29 5 ONT-206 81 0 ONT-207 37 4 ONT-20843 3 ONT-209 35 4 ONT-210 40 4

Stereorandom PS Oligonucleotides Having 2′-OMe-DNA-2′OMe (3-14-3)Design: (d Upper Case)=DNA; Lower Case=2′-OMe; s=Phosphorothioate

ONT-129 (SEQ ID NO: 571) cscscsd(TsCsTsGsGsAsTsTsGsAsGsCsAsTs)cscsaONT-130 (SEQ ID NO: 572) asasgsd(CsTsTsTsGsGsTsTsGsGsGsCsAsAs)csascONT-131 (SEQ ID NO: 573) asgstsd(CsAsCsTsTsGsGsGsAsGsCsTsTsCs)tscscONT-132 (SEQ ID NO: 574) csascsd(TsTsGsGsGsAsGsCsTsTsCsTsCsCs)tsgsgONT-133 (SEQ ID NO: 575) astsasd(GsCsCsAsTsTsGsCsAsGsCsTsGsCs)tscsaONT-134 (SEQ ID NO: 576) tsgsgsd(AsTsTsGsAsGsCsAsTsCsCsAsCsCs)asasgONT-135 (SEQ ID NO: 577) cscsasd(TsAsGsCsCsAsTsTsGsCsAsGsCsTs)gscstONT-136 (SEQ ID NO: 578) gstscsd(AsCsTsTsGsGsGsAsGsCsTsTsCsTs)cscstONT-137 (SEQ ID NO: 579) cscsasd(GsGsGsCsAsCsTsCsAsTsCsTsGsCs)astsgONT-138 (SEQ ID NO: 580) gscscsd(AsTsCsCsAsAsGsTsCsAsCsTsTsGs)gsgsaONT-139 (SEQ ID NO: 581) gsasasd(GsCsTsTsTsGsGsTsTsGsGsGsCsAs)ascsaONT-140 (SEQ ID NO: 582) cstsgsd(GsAsTsTsGsAsGsCsAsTsCsCsAsCs)csasaONT-155 (SEQ ID NO: 583) csasasd(GsTsCsAsCsTsTsGsGsGsAsGsCsTs)tscstONT-156 (SEQ ID NO: 584) astsgsd(CsCsAsTsCsCsAsAsGsTsCsAsCsTs)tsgsgONT-157 (SEQ ID NO: 585) astsgsd(AsGsAsTsGsCsCsTsGsGsCsTsGsCs)csastONT-158 (SEQ ID NO: 586) tstsgsd(GsGsAsGsCsTsTsCsTsCsCsTsGsGs)tsgsgONT-159 (SEQ ID NO: 587) tsgsgsd(GsAsGsCsTsTsCsTsCsCsTsGsGsTs)gsgsaONT-160 (SEQ ID NO: 588) tstsasd(TsGsAsGsAsTsGsCsCsTsGsGsCsTs)gscscONT-161 (SEQ ID NO: 589) gststsd(AsTsGsAsGsAsTsGsCsCsTsGsGsCs)tsgscONT-162 (SEQ ID NO: 590) cscsasd(AsGsTsCsAsCsTsTsGsGsGsAsGsCs)tstscONT-163 (SEQ ID NO: 591) asgscsd(TsTsTsGsGsTsTsGsGsGsCsAsAsCs)ascsaONT-164 (SEQ ID NO: 592) tsastsd(GsAsGsAsTsGsCsCsTsGsGsCsTsGs)cscsaONT-165 (SEQ ID NO: 593) tsgstsd(TsAsTsGsAsGsAsTsGsCsCsTsGsGs)cstsgONT-166 (SEQ ID NO: 594) astscsd(CsAsAsGsTsCsAsCsTsTsGsGsGsAs)gscstONT-167 (SEQ ID NO: 595) gsgsgsd(AsAsGsCsTsTsTsGsGsTsTsGsGsGs)csasaONT-168 (SEQ ID NO: 596) cstscsd(CsAsTsCsCsAsTsGsAsGsGsTsCsAs)tstscONT-169 (SEQ ID NO: 597) asasgsd(TsCsAsCsTsTsGsGsGsAsGsCsTsTs)cstscONT-170 (SEQ ID NO: 598) cscsasd(TsCsCsAsAsGsTsCsAsCsTsTsGsGs)gsasgONT-171 (SEQ ID NO: 599) tscscsd(AsAsGsTsCsAsCsTsTsGsGsGsAsGs)cststONT-172 (SEQ ID NO: 600) cscstsd(CsTsGsGsAsTsTsGsAsGsCsAsTsCs)csascONT-173 (SEQ ID NO: 601) ascstsd(TsGsGsGsAsGsCsTsTsCsTsCsCsTs)gsgstONT-174 (SEQ ID NO: 602) cststsd(GsGsGsAsGsCsTsTsCsTsCsCsTsGs)gstsgONT-175 (SEQ ID NO: 603) csastsd(GsCsCsAsTsCsCsAsAsGsTsCsAsCs)tstsgONT-176 (SEQ ID NO: 604) tsgscsd(CsAsTsCsCsAsAsGsTsCsAsCsTsTs)gsgsgONT-177 (SEQ ID NO: 605) tscscsd(AsTsCsCsAsTsGsAsGsGsTsCsAsTs)tscscONT-178 (SEQ ID NO: 606) asgsgsd(GsCsAsCsTsCsAsTsCsTsGsCsAsTs)gsgsgONT-179 (SEQ ID NO: 607) cscsasd(GsTsTsCsCsTsTsCsAsTsTsCsTsGs)csascONT-180 (SEQ ID NO: 608) csastsd(AsGsCsCsAsTsTsGsCsAsGsCsTsGs)cstscONT-181 (SEQ ID NO: 609) tscstsd(GsGsAsTsTsGsAsGsCsAsTsCsCsAs)cscsaONT-182 (SEQ ID NO: 610) gsgsasd(TsTsGsAsGsCsAsTsCsCsAsCsCsAs)asgsa

Biology In Vitro Data in HepG2 Cells for the 2′-OMe-DNA-2′-OMe (3-14-3)Design

FOXO1 Levels at 20 nM (%) SD ONT-129 82 5 ONT-130 49 4 ONT-131 92 3ONT-132 91 2 ONT-133 58 3 ONT-134 73 2 ONT-135 65 5 ONT-136 92 2 ONT-13794 2 ONT-138 78 1 ONT-139 61 1 ONT-140 82 4 ONT-155 95 2 ONT-156 74 1ONT-157 56 2 ONT-158 93 1 ONT-159 94 1 ONT-160 71 1 ONT-161 67 1 ONT-16289 1 ONT-163 55 7 ONT-164 68 4 ONT-165 70 1 ONT-166 89 4 ONT-167 81 0ONT-168 81 0 ONT-169 94 0 ONT-170 88 1 ONT-171 92 4 ONT-172 86 2 ONT-17390 1 ONT-174 93 2 ONT-175 84 1 ONT-176 80 2 ONT-177 83 2 ONT-178 95 2ONT-179 93 8 ONT-180 68 7 ONT-181 85 5 ONT-182 80 5 Levels at 200 nM (%)SD ONT-129 27 1 ONT-130 46 4 ONT-131 53 9 ONT-132 53 2 ONT-133 48 6ONT-134 35 9 ONT-135 45 15 ONT-136 40 7 ONT-137 50 4 ONT-138 80 3ONT-139 40 3 ONT-140 33 13 ONT-155 52 2 ONT-156 35 4 ONT-157 39 2ONT-158 87 6 ONT-159 89 5 ONT-160 33 10 ONT-161 40 11 ONT-162 60 7ONT-163 42 8 ONT-164 34 10 ONT-165 38 1 ONT-166 62 9 ONT-167 64 1ONT-168 38 2 ONT-169 67 3 ONT-170 74 8 ONT-171 65 5 ONT-172 33 18ONT-173 72 15 ONT-174 65 15 ONT-175 38 21 ONT-176 48 8 ONT-177 28 5ONT-178 97 11 ONT-179 47 6 ONT-180 56 12 ONT-181 45 26 ONT-182 33 17

Hit Selection:

ONT-151 (SEQ ID NO: 611) d(GsAsAsGsCsTsTs)tsgsgststsgsd(GsGsCsAsAsCsA)ONT-198 (SEQ ID NO: 612) d(CsCsAsTsCsCsAs)asgstscsascsd(TsTsGsGsGsAsG)ONT-185 (SEQ ID NO: 613) d(AsTsGsAsGsAsTs)gscscstsgsgsd(CsTsGsCsCsAsT)ONT-142 (SEQ ID NO: 614) d(AsAsGsCsTsTsTs)gsgststsgsgsd(GsCsAsAsCsAsC)ONT-145 (SEQ ID NO: 615) d(AsTsAsGsCsCsAs)tstsgscsasgsd(CsTsGsCsTsCsA)ONT-192 (SEQ ID NO: 616) d(TsAsTsGsAsGsAs)tsgscscstsgsd(GsCsTsGsCsCsA)ONT-188 (SEQ ID NO: 617) d(TsTsAsTsGsAsGs)astsgscscstsd(GsGsCsTsGsCsC)

Secondary Screen. Chemistry and Stereochemistry Screen Summary forOligonucleotide Synthesis on a DNA/RNA Synthesizer MerMade-12(Stereodefined Phosphorothioate 2′-Deoxy and 2′-OMe Cycle)

delivery wait time step reaction reagent vol (mL) (sec) 1 detritylation3% TCA in DCM 4 × 1   N.A. 2 coupling 0.15 M chiral 2 × 0.5 2 × 450phosphoramidite in (2′-OMe ACN + 2 M CMPT in RNA) ACN 2 × 300 (DNA) 3capping 1 5% Pac20 in THF/2,6- 1 60  lutidine 4 capping 2 5% Pac20 inTHF/2,6- 1 60  lutidine + 16% NMI in THF 5 sulfurization 0.3 MS-(2-cyanoethyl) 1 600 methylthiosulfonate

in ACN/BSTFA

Examples Applied on the FOXO1 Hit Sequences: Examples Include but arenot Limited to:

(SEQ ID NO: 618)(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 619)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 620)(Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 621)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 622)(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 623)(Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 624)(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 625)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 626)(Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 627)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 628)(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 629)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(OMe)d[TsTsGsGsGsAsG](SEQ ID NO: 630)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs)_(OMe) d[CsTsGsCsCsAsT](SEQ ID NO: 631)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(LNA)d[TsTsGsGsGsAsG](SEQ ID NO: 632)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs)_(LNA) d[CsTsGsCsCsAsT](SEQ ID NO: 633)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAs](AsGsTsCsAsCs) _(MOE)d[TsTsGsGsGsAsG](SEQ ID NO: 634)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTs](GsCsCsTsGsGs)_(MOE) d[CsTsGsCsCsAsT](SEQ ID NO: 635)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp,Sp)(CsCsAs)_(OMe)d[TsCsCsAsAsGsTsCsAsCsTsTsGsGs](GsAsG)_(OMe)(SEQ ID NO: 636)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp,Sp)(AsTsGs)_(MOE)d[AsGsAsTsGsCsCsTsGsGsCsTsGsCs](CsAsT)_(MOE)(SEQ ID NO: 637)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(CsCsAs)_(LNA)d[TsCsCsAsAsGsTsCsAsCsTsTsGsGs](GsAsG)_(LNA)(SEQ ID NO: 638)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(AsTsGs)_(OMe)d[AsGsAsTsGsCsCsTsGsGsCsTsGsCs](CsAsT)_(OMe)(SEQ ID NO: 639)(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 640)(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 641)(Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 642)(Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 643)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 644)(Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 645)(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp)d[GsAsAsGsCsTsTsTsGsGsTsTsGsGsGsCsAsAsCsA] (SEQ ID NO: 646)(Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Rp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 647)(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 648)(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 649)(Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp,Sp)d[AsTsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsT] (SEQ ID NO: 650)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 651)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 652)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 653)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 654)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 655)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)d[CsCsAsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGsAsG] (SEQ ID NO: 656)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp,Sp)(CsCs)_(OMe)d[AsTsCsCsAsAs](GsTsCs)_(OMe)d[AsCsTsTsGsGsGs](AsG)_(OMe)(SEQ ID NO: 657)(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp)(CsCs)_(LNA)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(LNA)(SEQ ID NO: 658)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp)(CsCs)_(MOE)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(MOE)(SEQ ID NO: 659)(Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp)(CsCs)_(OMe)d[AsTsCsCsAsAsGsTsCsAsCsTsTsGsGsGs](AsG)_(OMe)

Example 4. Suppression of Nucleic Acid Polymer

Among other things, the present disclosure provides chirally controlledoligonucleotide compositions and methods thereof that deliver unexpectedresults when, e.g., used for suppressing nucleic acid polymers through,in some cases, cleavage of such nucleic acid polymers. Examples includebut are not limited to those presented herein.

RNase H Assay

Cleavage rate of nucleic acid polymers by nucleases, for example, RNA byRNase H, is important with respect to the use of oligonucleotides intherapeutic technologies such as antisense technology. Using our assay,we investigated the cleavage rates and analyzed the metabolites forchirally controlled oligonucleotide compositions of particularoligonucleotide types (P-diastereomers) when oligonucleotides of theparticular oligonucleotide types are bound to complementary RNA. Resultsbelow also illustrate the importance of cleavage patterns recognized bythe present disclosure.

RNase H used herein is a ubiquitously expressed endonuclease thathydrolyses the RNA strand of a RNA/DNA hybrid. It plays an importantrole in the mode of action of antisense oligonucleotides. In someembodiments, RNase H cleavage rate is significantly reduced when the RNAsubstrate is structured (Lima, W. F., Venkatraman, M., Crooke, S. T. TheInfluence of Antisense Oligonucleotide-induced RNA Structure onEscherichia coli RNase H1 Activity The Journal Of Biological Chemistry272, No. 29, 18191-18199, (1997)). Furthermore, the 2′-MOE gapmerdesigns (5-10-5) offer higher affinities for RNA targets leading tominimal turnover of the antisense strand. Presence of 2′-MOEmodifications in the wings also reduce the number of RNase H cleavagesites.

To study the RNA cleavage rate, the present disclosure provides a simpleassay to quantify the length of RNA remaining after incubation withRNase H. The provided method, among other things, provides the relativerates of RNase H cleavage for stereorandom 2′-modified gapmers,stereorandom DNA oligonucleotide compositions and chirally pureP-diastereomers (chirally controlled oligonucleotide compositions of acorresponding oligonucleotide type) for various oligomers for differenttargets. Changing the stereochemistry at 2′-modified regions and the DNAcore provides information with respect to how stereochemistry in theseregions affects the interaction of RNase H to its substrates. RNase Hreaction mixtures at different time points were analyzed by LCMS todetermine the cleavage pattern. The present disclosure, among otherthings, provides nucleic acid polymer, for example RNA, cleavage ratesand cleavage patterns (maps) that are critical to design stereochemicalnucleic acid architectures for optimal activity, e.g., antisenseactivity.

Equipment:

Alliance HPLC, 2489—TUV, 2695E—Equipped with autosampler

Caryl00 (Agilent Technologies)

Methods:

DNA/RNA Duplex Preparation:

Oligonucleotide concentrations were determined by measuring theabsorbance in water at 260 nm. DNA/RNA duplexes were prepared by mixingequimolar solutions oligonucleotides with each strand concentration of10 uM. The mixtures were heated at 90° C. for 2 minutes in water bathand were cooled down slowly over several hours.

Human RNase H Protein Expression and Purification:

Human RNase HC clone was obtained from Prof Wei Yang's laboratory at NIHBethesda. The protocol for obtaining this human RNase HC (residues136-286) has been described (Nowotny, M. et al. Structure of Human RNaseH1 Complexed with an RNA/DNA Hybrid: Insight into HIV ReverseTranscription. Molecular Cell 28, 264-276, (2007). The proteinexpression was carried out by following reported protocol with theexception that the resulting protein had an N-terminal His6 tag (SEQ IDNO: 1552). BL21(DE3) E. coli cells in LB medium were used for proteinexpression. Cells were grown at 37° C. till OD600 reached around 0.7.The cultures were then cooled and 0.4 mM IPTG was added to induceprotein expression overnight at 16° C. E. coli extract was prepared bysonication in buffer A (40 mM NaH₂PO₄ (pH 7.0), 1 M NaCl, 5% glycerol,2.8 mM β-mercaptoethanol and 10 mM imidazole) with the addition ofprotease inhibitors (Sigma-Aldrich). The extract was purified by Niaffinity column using buffer A plus 60 mM imidazole. The protein waseluted with a linear gradient of 60 to 300 mM imidazole. The proteinpeak was collected and was further purified on a Mono S column (GEHealthcare) with a 100 mM-500 mM gradient of NaCl in buffer B. Fractionscontaining RNase HC were concentrated to 0.3 mg/ml in the storage buffer(20 mM HEPES (pH 7.0), 100 mM NaCl, 5% glycerol, 0.5 mM EDTA, 2 mM DTT)and stored at −20° C. 0.3 mg/ml enzyme concentration corresponds to 17.4uM based on its reported extinction coefficient (32095 cm⁻¹ M⁻¹) and MW(18963.3 Da units).

RNase H Assay:

In a 96-well plate, to 25 μL DNA/RNA duplex (10 μM) was added 5 μL of10×RNase H buffer followed by 15 μL water. The mixture was incubated at37° C. for a few minutes and then 5 μL of 0.1 μM stock solution ofenzyme was added to give total volume of 50 μL with finalsubstrate/enzyme concentration 5 μM/0.01 μM (500:1) and was furtherincubated at 37° C. Various ratios of the DNA/RNA duplex: RNase Hprotein were studied using these conditions to find an optimal ratio tostudy the kinetics. The reactions were quenched at different time pointsusing 10 μL of 500 mM EDTA disodium solution in water. For zero min timepoint, EDTA was added to the reaction mixture before the addition ofenzyme. Controls were run to ensure that EDTA was able to successfullyinhibit the enzyme activity completely. After all the reactions werequenched 10 μL of each reaction mixture was injected on to analyticalHPLC column (XBridge C18, 3.5 um, 4.6×150 mm, Waters Part #186003034).Kcat/Km can be measured by a number of methods, such as FRET(Fluorescence Resonance Energy Transfer) dependent RNase H assay usingdual labeled RNA and monitored by SpectraMax.

Solid Phase Extraction Protocol for Sample Preparation for LCMS:

96 well plate (Waters part #186002321) was used to clean the RNase Hreaction mixture before running LCMS. 500 μL of acetonitrile followed bywater was used to equilibrate the plate under mild vacuum with the helpof manifold (Millipore part # MSV MHTS00). Precaution was taken not tolet the plate dry. About 50-100 μL of RNase H reaction mixture wasloaded in each well followed by water washings (2 mL) under mild vacuum.2×500 μL of 70% ACN/Water was used to recover the sample. The recoveredsamples were transferred to 2 mL centrifuge tubes and were concentratedto dryness in speed vac. Each dry sample was reconstituted in 100 μLwater and 10 μL was injected on Acquity UPLC@OST C18 1.7 um, 2.1×50 mm(part #186003949) for LCMS analysis.

For mass spectrometry analysis, the reaction mixtures after quenchingwere cleaned using Cis 96 well plate (Waters). The oligomers were elutedin 70% Acetonitrile/Water. The Acetonitrile was evaporated usingspeedvac and the resulting residue was reconstituted in water forinjection.

Eluent A=50 mM Triethyl ammonium acetate

Eluent B=Acetonitrile Column Temperature=60° C.

UV was recorded at 254 nm and 280 nm

RP-HPLC Gradient Method

Time (min) Flow (ml/min) % A % B Curve 1 0.0 1.00 95.0 5.0 2 2.00 1.0095.0 5.0 1 3 22.00 1.00 80.0 20.0 6 4 25.00 1.00 5.0 95.0 6 5 25.5 1.0095.0 5.0 1 6 30 1.00 95.0 5.0 1

On HPLC chromatograms, peak areas corresponding to full length RNAoligomer (ONT-28) were integrated, normalized using the DNA peak andwere plotted against time (FIG. 8). ONT-87 demonstrated superiorcleavage for complementary RNA when in duplex form, in comparison to theother product candidates and Mipomersen. Since all the diastereomers inthis panel have 2′-MOE modified wings that do not activate RNase Henzyme, without the intention to be limited by theory, Applicant notesthat activity is likely dictated by the stereochemistry in the DNA core.Heteroduplexes with ONT-77 to ONT-81 including Mipomersen in theantisense strand show very similar RNA cleavage rates. ONT-89 withalternating Sp/Rp stereochemistry showed the least activity in thetested time frame under the tested conditions. Among the testedoligonucleotides with MOE modifications, ONT-87 and ONT-88 units in theantisense strand exhibited increased in activity in comparison to therest of the heteroduplexes. Particularly, ONT-87 provided surprisinglyhigh cleavage rate and unexpected low level of remaining target RNA.Additional example data were illustrated in FIG. 6 and FIG. 24.

In Vitro Oligonucleotide Transfection Assay:

Transfection assays are widely known and practiced by persons havingordinary skill in the art. An example protocol is described herein.Hep3B cells are reverse transfected with Lipofectamine 2000 (LifeTechnologies, Cat. No. 11668-019) at 18×10³ cells/well density in96-well plates using the manufacturer's protocol. For dose responsecurves eight ⅓ serial dilutions are used starting from 60-100 nM. 25 μLof 6× oligonucleotide concentration is mixed with a prepared mixture of0.4 μL Lipofectamine 2000 with 25 μL of serum-free medium Opti-MEMmedium (Gibco, Cat. No. 31985-062) per well. After a 20 min minuteincubation, 100 μL of 180×10³ cells/ml suspended in 10% FBS in DMEM cellculture media (Gibco, Cat. No. 11965-092) is added to bring the finalvolume to 150 μL per well. 24-48 hours post transfection Hep3B cells arelysed by adding 75 μL of Lysis Mixture with 0.5 mg/ml Proteinase K usingQuantiGene Sample Processing Kit for Cultured Cells (Affymetrix, Cat.No. QS0103). The Target mRNA and GAPDH mRNA expression levels in celllysates are measured using Affymetrix QuantiGene 2.0 Assay Kit (Cat. No.QS0011) according to the manufacturer's protocol. The Target mRNAexpression is normalized to GAPDH mRNA expression from the same sample;and relative Target/GAPDH levels are compared to transfections usingLipofectamine 2000 only (no oligonucleotide) control. Dose responsecurves are generated by GraphPad Prism 6 using nonlinear regression log(inhibitor) vs. response curve fit with variable slope (4 parameters).For example results, see FIG. 24, FIG. 27 and FIG. 29.

Example 5. Provided Compositions and Methods Provide Control of CleavagePatterns

The present disclosure surprisingly found that internucleotidic linkagestereochemistry pattern has unexpected impact on cleavage patterns ofnucleic acid polymers. By changing common patterns of backbone chiralcenters of chirally controlled oligonucleotide compositions, numbers ofcleavage sites, cleavage percentage at a cleavage site, and/or locationsof cleavage sits can be surprisingly altered, both independently and incombination. As described in the example herein, provided compositionsand methods can provide control over cleavage patterns of nucleic acidpolymers.

Using similar assay conditions, various chirally controlledoligonucleotide compositions of different oligonucleotide types weretested. Example cleavage patterns of the target RNA sequence ispresented in FIG. 9. Certain pattern of backbone chiral centers, such asthat in ONT-87 and ONT-154, surprisingly produces only one cleavage sitein the target sequence. Moreover, it is surprisingly found thatoligonucleotides providing single cleavage site, such as ONT-87 andONT-154, provide unexpectedly high cleavage rate and low level ofremaining target nucleic acid polymer. See also FIG. 8, FIG. 10 and FIG.11.

Example 6. Example Cleavage of FOXO1 mRNA

Oligonucleotide compositions targeting different regions of FOXO1 mRNAwere tested in cleavage assays as described above. In each case,chirally controlled oligonucleotide compositions were shown to becapable of providing altered cleavage patterns relative to referencecleavage patterns from chirally uncontrolled oligonucleotidecompositions sharing the same common base sequence and length. Forexample results, see FIG. 10 and FIG. 11. As shown in FIG. 12, examplechirally controlled oligonucleotide compositions provide bothsignificantly faster cleavage rates and unexpectedly low levels ofremaining substrates when compared to reference chirally uncontrolledoligonucleotide compositions. In some embodiments, as shown in FIG. 11,the cleavage sites are associated with RpSpSp backbone chiral centersequence. In some embodiments, cleavage sites are two base pairsupstream of RpSpSp.

Example oligonucleotide compositions are listed below.

Tm SEQ Oligo Sequence Description (° C.) ID NO: ONT-366dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdTsd All DNA 66.5 660GsdCsdCsdAsdTsdA ONT-389 dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdTsdS₇RSSRSSR 64.3 661 GsdCsdCsdAsdTsdA S₅ ONT-390dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdTsd S₆RSSRSSR 64.6 662GsdCsdCsdAsdTsdA S₆ ONT-391 dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdTsdS₅RSSRSSR 64.3 663 GsdCsdCsdAsdTsdA S₇ ONT-387rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCrUrCrA complementary 664 RNA ONT-367dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdTsd All DNA 62.9 665GsdCsdTsdCsdAsdC ONT-392 dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdTsdS₇RSSRSSR 59.5 666 GsdCsdTsdCsdAsdC S₅ ONT-393dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdTsd S₆RSSRSSR 60 667GsdCsdTsdCsdAsdC S₆ ONT-394 dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdTsdS₅RSSRSSR 59.5 668 GsdCsdTsdCsdAsdC S₇ ONT-388rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGrCrUrA complementary 669 RNA

Example 7. Example Chirally Controlled Oligonucleotide CompositionsProvide Higher Turn-Over

In cases where the Tm of cleaved nucleic acid polymer fragments, forexample RNA fragments, to oligonucleotides is greater than aphysiological temperature, product dissociation may be inhibited andoligonucleotides may not be able to dissociate and find other targetstrands to form duplexes and cause the target strands to be cleaved. TheTm of ONT-316 (5-10-5 2′-MOE Gapmer) to complementary RNA is 76° C.After a cut or a few cuts in the RNA sequence complementary to theoligonucleotides, the 2′-MOE fragments may remain bound to RNA and thuscannot cause the other target molecules to be cleaved. Thermal meltingtemperatures of DNA strands generally are much lower when duplexed toRNA, for example, ONT-367 (63° C.) and ONT-392 60° C.). Additionally,thermal stability in DNA sequences is often relatively uniformlydistributed compared to 2′-MOE modified oligonucleotides. In someembodiments, oligonucleotides in provided chirally controlledoligonucleotide compositions do not contain 2′-modifications such as2′-MOE. In some embodiments, oligonucleotides in provided chirallycontrolled oligonucleotide compositions, which do not contain2′-modifications such as 2′-MOE, more easily dissociate from nucleicacid polymer cleavage fragments, and have higher turn-over thanoligonucleotides having 2′-modifications such as 2′-MOE. In someembodiments, the present disclosure provides an all DNA designs, inwhich oligonucleotides do not have 2′-modifications. In someembodiments, chirally controlled oligonucleotide compositions whereinoligonucleotides having no 2′-modification provides higher turn-over ofa nuclease such as RNase H. In some embodiments, after cleavage RNase Hdissociates more easily from duplex formed by RNA and oligonucleotidesof provided chirally controlled oligonucleotide compositions. Usingsimilar protocols as described above, turn-over of two example chirallycontrolled oligonucleotide compositions of oligonucleotide type ONT-367and ONT-392 indeed showed higher turn-over rate than reference chirallyuncontrolled oligonucleotide compositions (see FIG. 13).

Example 8. Example Cleavage of FOXO1 mRNA

As exemplified in FIG. 14, chirally controlled oligonucleotidecompositions and methods thereof in the present disclosure can providecontrolled cleavage of nucleic acid polymers. In some embodiments,chirally controlled oligonucleotide compositions of the presentdisclosure produces altered cleavage pattern in terms of number ofcleavage sites, location of cleavage sites, and/or relative cleavagepercentage of cleavage sites. In some embodiments, as exemplified byONT-401 and ONT-406, chirally controlled oligonucleotide compositionsprovide single site cleavage.

In some embodiments, only one component from RNA cleavage was detected.Without the intention to be limited by theory, Applicant notes that suchobservation could be due to the processive nature of RNase H enzymewhich could make multiple cuts on the same duplex resulting in muchshorter 5′-OH 3′-OH fragments.

Additional chirally controlled oligonucleotide compositions were furthertested. As described above, provided chirally controlled oligonucleotidecompositions provides unexpected results, for example, in terms ofcleavage rate and % RNA remaining in DNA/RNA duplex. See FIGS. 15-17.Example analytical data were presented in FIGS. 18-20. Without theintention to be limited by theory, Applicant notes that in someembodiments, cleavage may happen as depicted in FIG. 21. In FIG. 17, itis noted ONT-406 was observed to elicit cleavage of duplexed RNA at arate in slight excess of that of the natural DNA oligonucleotide ONT-415having the same base sequence and length. Applicant notes that chirallycontrolled oligonucleotide compositions of ONT-406, and other chirallycontrolled oligonucleotide compositions provided in this disclosure,have other preferred properties that an ONT-415 composition does nothave, for example, better stability profiles in vitro and/or in vivo.Additional example data were presented in FIG. 25. Also, as will beappreciated by those skilled in the art, example data illustrated inFIG. 26 and FIG. 27 confirm that provided example chirally controlledoligonucleotide compositions, especially when so designed to control thecleavage patterns through patterns of backbone chiral centers, producedmuch better results than reference oligonucleotide compositions, e.g., astereorandom oligonucleotide composition. As exemplified in FIG. 26,controlled patterns of backbone chiral centers, among other things, canselectively increase and/or decrease cleavage at existing cleavage sitewhen a DNA oligonucleotide is used, or creates entirely new cleavagesites that do not exist when a DNA oligonucleotide is used (see FIG. 25,ONT-415). In some embodiments, cleavage sites from a DNA oligonucleotideindicate endogenous cleavage preference of RNase H. As confirmed by FIG.27, provided chirally controlled oligonucleotide compositions arecapable of modulating target cleavage rate. In some embodiments,approximately 75% of the variance in cellular activity is accounted forby differences in cleavage rate which can be controlled through patternsof backbone chiral centers. As provided in this Application, furtherstructural features such as base modifications and their patterns, sugarmodification and their patterns, internucleotidic linkage modificationsand their patterns, and/or any combinations thereof, can be combinedwith patterns of backbone chiral centers to provide desiredoligonucleotide properties.

Example 9. Example Allele-Specific Suppression of mHTT

In some embodiments, the present disclosure provides chirally controlledoligonucleotide compositions and methods thereof for allele-specificsuppression of transcripts from one particular allele with selectivityover the others. In some embodiments, the present disclosure providesallele-specific suppression of mHTT.

FIG. 22 illustrates example chirally controlled oligonucleotidecompositions that specifically suppress transcripts from one allele butnot the others. Oligonucleotides 451 and 452 were tested withtranscripts from both exemplified alleles using biochemical assaysdescribed above. Allele-specific suppression is also tested in cells andanimal models using similar procedures as described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; US patent application publication US2013/0197061; and Østergaard et al., Nucleic Acids Research, 2013,41(21), 9634-9650. In all cases, transcripts from the target allele areselectively suppressed over those from the other alleles. As will beappreciated by those skilled in the art, example data illustrated inFIG. 22 confirm that provided example chirally controlledoligonucleotide compositions, especially when so designed to control thecleavage patterns through stereochemistry, produced much better resultsthan reference oligonucleotide compositions, in this case, astereorandom oligonucleotide composition. As confirmed by FIG. 22,patterns of backbone chiral centers can dramatically change cleavagepatterns (FIG. 22 C-E), and stereochemistry patterns can be employed toposition cleavage site at the mismatch site(FIG. 22 C-E), and/or candramatically improve selectivity between the mutant and wild type (FIG.22 G-H). In some embodiments, chirally controlled oligonucleotidecompositions are incubated with wtRNA and muRNA of a target and both theduplexes are incubated with RNase H.

Huntingtin Allele Tm

Mutant Huntingtin Allele ONT-453/ONT-451 38.8° C. Wild Type HuntingtinAllele ONT-454/ONT-451 37.3° C. Mutant Huntingtin Allele ONT-453/ONT-45238.8° C. Wild Type Huntingtin Allele ONT-454/ONT-452 36.5° C. MutantHuntingtin Allele ONT-453/ONT-450 40.3° C. Wild Type Huntingtin AlleleONT-454/ONT-450 38.8° C.

Example 10. Example Allele-Specific Suppression of FOXO1

In some embodiments, the present disclosure provides allele-specificsuppression of FOXO1.

FIG. 23 illustrates example chirally controlled oligonucleotidecompositions that specifically suppress transcripts from one allele butnot the others. Oligonucleotides ONT-400, ONT-402 and ONT-406 weretested with transcripts from both exemplified alleles using biochemicalassays described above. Allele-specific suppression is also tested incells and animal models using similar procedures as described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; US patent application publication US2013/0197061; Østergaard et al., Nucleic Acids Research 2013, 41(21),9634-9650; and Jiang et al., Science 2013, 342, 111-114. Transcriptsfrom the target allele are selectively suppressed over those from theother alleles. In some cases, two RNAs with mismatch ONT-442 (A/G,position 7th) and ONT-443 (A/G, position 13^(th)) from ONT-388 aresynthesized and are duplexed with ONT-396 to ONT-414. RNase H assay areperformed to obtain cleavage rates and cleavage maps.

Example 11. Certain Example Oligonucleotides and OligonucleotideCompositions

Stereorandom oligonucleotides with different 2′ substitution chemistriestargeting three distinct regions of FOXO1 mRNA with the thermal meltingtemperatures when duplexed with complementary RNA. The concentration ofeach strand was 1 uM in 1×PBS buffer.

SEQ Oligo Sequence Description Tm (° C.) ID NO: ONT-316TeosAeosGeos5mCeos5mCeosdAsdTsdTsdGs5md 5-10-5 (2′-MOE 76.7 670CsdAsdGs5mdCsdTsdGs5mCeosTeos5mCeosAeos Gapmer) 5mCeo ONT-355dTsdAsdGsdCsdCsdAsdTstsgscsasgscsdTsdGsdCs 7-6-7 (DNA-2′- 71.2 671dTsdCsdAsdC OMe-DNA) Gapmer ONT-361tsasgsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsdTsdG 3-14-3 (2′-OMe- 65.8 672sdCsdTscsascs DNA-2′-OMe) Gapmer ONT-367dTsdAsdGsdCsdCsdAsdTsdTsdGsdCsdAsdGsdCsd All DNA 62.9 673TsdGsdCsdTsdCsdAsdC ONT-373 tsasgscscsdAsdTsdTsdGsdCsdAsdGsdCsdTsdGscst5-10-5 (2′-OMe 71.8 674 scsasc Gapmer) ONT-388rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGrCrUrA Complementary 675 RNA ONT-302Teos5mCeos5mCeosAeosGeosdTsdTs5mdCs5mdC 5-10-5 (2′-MOE 72.5 676sdTsdTs5mdCsdAsdTsdTs5mCeosTeosGeos5mCe Gapmer) osAeo ONT-352dTsdCsdCsdAsdGsdTsdTscscststscsasdTsdTsdCsd 7-6-7 (DNA-2′- 65.4 677TsdGsdCsdA OMe-DNA) Gapmer ONT-358tscscsdAsdGsdTsdTsdCsdCsdTsdTsdCsdAsdTsdTs 3-14-3 (2′-OMe- 62.6 678dCsdTsgscsas DNA-2′-OMe) Gapmer ONT-364dTsdCsdCsdAsdGsdTsdTsdCsdCsdTsdTsdCsdAsd All DNA 58.4 679TsdTsdCsdTsdGsdCsdA ONT-370 tscscsasgsdTsdTsdCsdCsdTsdTsdCsdAsdTsdTscsts5-10-5 (2′-OMe 68 680 gscsa Gapmer) ONT-386rUrGrCrArGrArArUrGrArArGrGrArArCrUrGrGrA Complementary 681 RNA ONT-315TeosGeosAeosGeosAeosdTsdGs5mdCs5mdCsdTs 5-10-5 (2′-MOE 77.5 682dGsdGs5mdCsdTsdGs5mCeos5mCeosAeosTeosAeo Gapmer) ONT-354dTsdGsdAsdGsdAsdTsdGscscstsgsgscsdTsdGsdCs 7-6-7 (DNA-2′- 75.5 683dCsdAsdTsdA OMe-DNA) Gapmer ONT-360tsgsasdGsdAsdTsdGsdCsdCsdTsdGsdGsdCsdTsdG 3-14-3 (2′-OMe- 69 684sdCsdCsastsas DNA-2′-OMe) Gapmer ONT-366dTsdGsdAsdGsdAsdTsdGsdCsdCsdTsdGsdGsdCs All DNA 66.5 685dTsdGsdCsdCsdAsdTsdA ONT-372 tsgsasgsasdTsdGsdCsdCsdTsdGsdGsdCsdTsdGscs5-10-5 (2′-OMe 74.4 686 csastsa Gapmer) ONT-387rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCrUrCrA Complementary 687 RNA

Additional example stereorandom oligonucleotide compositions are listedbelow.

SEQ ID Oligo Sequence (5′ to 3′) NO: ONT-41(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs 6885mCsAs5mCs5mC)_(MOE) ONT-70(Gs5mCs)_(MOE)d[GsTsTsTsGs5mCsTs5mCsTsTs5mCsTsTs](5mCsTsT 689sGs5mCGs)_(MOE)d[TsTsTsTs](TsT)_(MOE) ONT-83(GsTs5mCs5mCs5mCs)_(MOE)d(TsGsAsAsGsAsTsGsTs5mCs](AsAsTs 690Gs5mC)_(MOE) ONT-302(Ts5mCs5mCsAsGs)_(MOE)d[TsTs5mCs5mCsTsTs5mCsAsTsTs](5mCs 691TsGs5mCsA)_(MOE) ONT-315(TsGsAsGsAs)_(MOE)d[TsGs5mCs5mCsTsGsGs5mCsTsGs](5mCs5mCs 692AsTsA)_(MOE) ONT-316 (TsAsGs5mCs5mCs)_(MOE)d[AsTsTsGs5mCsAsGs5mCsTsGs5m]693 (CsTs5mCsAs5mC)_(MOE) ONT-352[TsCsCsAsGsTsTs](cscststscsas)_(OMe)d[TsTsCsTsGsCsA] 694 ONT-354[TsGsAsGsAsTsGs](CsCsTsGsGsCs)_(OMe)d[TsGsCsCsAsTsA] 695 ONT-355[TsAsGsCsCsAsTs](TsGsCsAsGsCs)_(OMe)d[TsGsCsTsCsAsC] 696 ONT-358(TsCsCs)_(OMe)d[AsGsTsTsCsCsTsTsCsAsTsTsCsTs](GsCsA)_(OMe) 697 ONT-360(TsGsAs)_(OMe)d[GsAsTsGsCsCsTsGsGsCsTsGsCsCs](AsTsA)_(OMe) 698 ONT-361(TsAsGs)_(OMe)d[CsCsAsTsTsGsCsAsGsCsTsGsCsTs](CsAsC)_(OMe) 699 ONT-364[TsCsCsAsGsTsTsCsCsTsTsCsAsTsTsCsTsGsCsA] 700 ONT-366[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA] 701 ONT-367[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 702 ONT-370(TsCsCsAsGs)_(OMe)d[TsTsCsCsTsTsCsAsTsTs](CsTsGsCsA)_(OMe) 703 ONT-372(TsGsAsGsAs)_(OMe)d[TsGsCsCsTsGsGsCsTsGs](CsCsAsTsA)_(OMe) 704 ONT-373(TsAsGsCsCs)_(OMe)d[AsTsTsGsCsAsGsCsTsGs](CsTsCsAsC)_(OMe) 705 ONT-440(UsAsGsCsCs)_(F)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 706 ONT-441(UsAsGsCsCs)_(F)d[AsTsTsGsCsAsGsCsTsGsC] 707 ONT-460(TsAsGsCsCs)_(OMe)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 708 ONT-450[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC] 709

Example RNA and DNA oligonucleotides are listed below.

SEQ ID Oligo Sequence (5′ to 3′) NO: ONT-28rGrGrUrGrCrGrArArGrCrArGrArCrUrGrAr 710 GrGrC ONT-386rUrGrCrArGrArArUrGrArArGrGrArArCrUr 711 GrGrA ONT-rUrArUrGrGrCrArGrCrCrArGrGrCrArUrCr 712 387 UrCrA ONT-rGrUrGrArGrCrArGrCrUrGrCrArArUrGrGr 713 388 CrUrA ONT-d[TAGCCATTGCAGCTGCTCAC] 714 415 ONT- rGrUrGrArGrCrGrGrCrUrGrCrArArUrGrGr715 442 CrUrA ONT- rGrUrGrArGrCrArGrCrUrGrCrGrArUrGrGr 716 443 CrUrAONT- rGrGrUrGrArUrGrArCrArArUrUrUrArUrUr 717 453 ArArU ONT-rGrGrUrGrArUrGrGrCrArArUrUrUrArUrUr 718 454 ArArU

Example chirally pure oligonucleotides are presented below. In someembodiments, the present disclosure provides corresponding chirallycontrolled oligonucleotide compositions of each of the following exampleoligonucleotides.

SEQ Oligo Stereochemistry/Sequence (5′ to 3′) Description ID NO: ONT-389(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, 7S-(RSS)₃- 719Rp, Sp, Sp, Sp, Sp, Sp)- 3S d[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA]ONT-390 (Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 6S-(RSS)₃-720 Sp, Sp, Sp, Sp, Sp, Sp)- 4Sd[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA] ONT-391(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 5S-(RSS)₃- 721Sp, Sp, Sp, Sp, Sp, Sp)- 5S d[TsGsAsGsAsTsGsCsCsTsGsGsCsTsGsCsCsAsTsA]ONT-392 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, 7S-(RSS)₃-722 Rp, Sp, Sp, Sp, Sp, Sp)- 3Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-393(Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 6S-(RSS)₃- 723Sp, Sp, Sp, Sp, Sp, Sp)- 4S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-394 (Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 5S-(RSS)₃-724 Sp, Sp, Sp, Sp, Sp, Sp)- 5Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-396(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 18S-1R 725Sp, Sp, Sp, Sp, Sp, Rp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-397 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 17S-RS 726Sp, Sp, Sp, Sp, Rp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-398 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 16S-(RSS)727 Sp, Sp, Sp, Rp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-399 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 15S-(RSS)-728 Sp, Sp, Rp, Sp, Sp, Sp)- 1Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-400(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 14S-(RSS)- 729Sp, Rp, Sp, Sp, Sp, Sp)- 2S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-401 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 13S-(RSS)-730 Rp, Sp, Sp, Sp, Sp, Sp)- 3Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-402(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, 12S-(RSS)- 731Sp, Sp, Sp, Sp, Sp, Sp)- 4S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-403 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, 11S-(RSS)-732 Sp, Sp, Sp, Sp, Sp, Sp)- 5Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-404(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, 10S-(RSS)- 733Sp, Sp, Sp, Sp, Sp, Sp)- 6S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-405 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, 9S-(RSS)-734 Sp, Sp, Sp, Sp, Sp, Sp)- 7Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-406(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, 8S-(RSS)- 735Sp, Sp, Sp, Sp, Sp, Sp)- 8S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-407 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, 7S-(RSS)-736 Sp, Sp, Sp, Sp, Sp, Sp)- 9Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-408(Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, 6S-(RSS)- 737Sp, Sp, Sp, Sp, Sp, Sp)- 10S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-409 (Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 5S-(RSS)-738 Sp, Sp, Sp, Sp, Sp, Sp)- 11Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-410(Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 4S-(RSS)- 739Sp, Sp, Sp, Sp, Sp, Sp)- 12S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-411 (Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 3S-(RSS)-740 Sp, Sp, Sp, Sp, Sp, Sp)- 13Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-412(Sp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 2S-(RSS)- 741Sp, Sp, Sp, Sp, Sp, Sp)- 14S d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-413 (Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, S-(RSS)-742 Sp, Sp, Sp, Sp, Sp, Sp)- 15Sd[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-414(Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, (RSS)-16S 743Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-421 All-(Sp)- All S 744 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-422 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, 8S-(RSS)-745 Sp, Rp, Sp, Sp, Sp, Sp)-C6-amino- 3S-(RSS)-d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 2S ONT-455 All-(Rp)- All R746 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-451(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 13S-(RSS)- 747Rp, Sp, Sp, Sp, Sp, Sp)- 3S d[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC]ONT-452 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 14S-(RSS)-748 Sp, Rp, Sp, Sp, Sp, Sp)- 2Sd[AsTsTsAsAsTsAsAsAsTsTsGsTsCsAsTsCsAsCsC] ONT-75 All-(Rp)- All R 749(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-76(Sp, Rp, Rp, Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, SRRSR- 750Sp, Rp, Sp, Rp)- 11S-RSR (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-77(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 5R-10S-4R 751Sp, Rp, Rp, Rp, Rp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-80 All-(Sp)- All S 752(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-81(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, 5S-10R-4S 753Rp, Sp, Sp, Sp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-82 All-(Rp)- All R 754(GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs 5mCs](AsAsTsGs5mC)_(MOE)ONT-84 All-(Sp)- All S 755 (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-85(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, 5R-10S-4R 756Sp, Rp, Rp, Rp, Rp)- (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-86(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, 5S-10R-4S 757Rp, Sp, Sp, Sp, Sp)- (GsTs5mCs5mCs5mCs)_(MOE)d[TsGsAsAsGsAsTsGsTs5mCs](AsAsTsGs5mC)_(MOE) ONT-87(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, 5R-2S- 758Rp, Rp, Rp, Rp, Rp)- (RSS)₂-6R(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-88(Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, 5S-(RRS)₃- 759Sp, Sp, Sp, Sp, Sp)- 5S (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-89(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, (SR)₉S 760Sp, Rp, Sp, Rp, Sp)- (Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCsTsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-154(Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 7S-(RSS)₃-761 Sp, Sp, Sp, Sp)- 3S d[Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC] ONT-75 All-(Rp)- All-R 762(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCs TsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE) ONT-80 All-(Sp)- All-S 763(Gs5mCs5mCsTs5mCs)_(MOE)d[AsGsTs5mCsTsGs5mCs TsTs5mCs](Gs5mCsAs5mCs5mC)_(MOE)

Additional example oligonucleotides targeting FOXO1 with Tm arepresented below. In some embodiments, the present disclosure providescorresponding chirally controlled oligonucleotide compositions of eachof the following example oligonucleotides.

SEQ ID Oligo Sequence (5′ to 3′) Tm (° C.) NO: ONT-439[UsAsGs]_(F)d[CsCsAsTsTsGsCsAsGsCsTsGsCsTs][CsAs 68.3 764 C]_(F) ONT-440[UsAsGsCsCs]_(F)d[AsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 70.0 765 ONT-441[UsAsGsCsCs]_(F)d[AsTsTsGsCsAsGsCsTsGsC] 65.5 766 ONT-455 All-(Rp)- 66.8767 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-316[TsAsGs5mCs5mCs]_(MOE)d[AsTsTsGs5mCsAsGs5mCsTs 76.9 768Gs][5mCsTs5mCsAs5mC]_(MOE) ONT-367d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] 62.8 769 ONT-415d[TAGCCATTGCAGCTGCTCAC] 72.6 770 ONT-416[TsAsGsCsCsAsTsTsGsCsAsGsCs]_(OMe)d[TsGsCsTsCsAs 78.4 771 C] ONT-421All-(Sp)- 59.2 772 d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC] ONT-394(Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, 60.0 773Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]ONT-406 (Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Sp, Sp, Sp, Sp, 58.5 774Sp, Sp, Sp, Sp, Sp, Sp)- d[TsAsGsCsCsAsTsTsGsCsAsGsCsTsGsCsTsCsAsC]

Example 12. Example Additional Controlled Cleavage by Provided ChirallyControlled Oligonucleotide Compositions

As will be appreciated by those skilled in the art, example dataillustrated in FIG. 26 confirm that provided chirally controlledoligonucleotide compositions and methods thereof provided unexpectedresults compared to reference compositions, such as stereorandomoligonucleotide compositions. Among other things, chirally controlledoligonucleotide compositions can produce controlled cleavage patterns,including but not limited to controlling of positions of cleavage sites,numbers of cleavage sites, and relative cleavage percentage of cleavagesites. See also example data presented in FIG. 27.

Example 13. Stability of Chirally Controlled OligonucleotideCompositions

As will be appreciated by those skilled in the art, example dataillustrated in FIG. 26 confirm that stability of provided chirallycontrolled oligonucleotide compositions can be adjusted by varyingpatterns of backbone chiral centers. For example data, see FIG. 7 andFIG. 28. An example protocol for performing serum stability experimentis described below.

Protocol:

P-stereochemically pure PS DNA (ONT-396-ONT-414 (single Rp walk from3′end to 5′end)), stereorandom PS DNA (ONT-367), all-Sp PS DNA (ONT-421)and all-Rp PS DNA (ONT-455) were incubated in Rat serum (Sigma, R9759)(0 h and 48 h) and analyzed by IEX-HPLC.

Incubation Method:

5 μL of 250 μM of each DNA solutions and 45 μL of Rat serum were mixedand incubated at 37° C. for each time points (0 h and 48 h). At eachtime points, reaction was stopped by adding 25 μL of 150 mM EDTAsolution, 30 μL of Lysis buffer (erpicentre, MTC096H) and 3 μL ofProteinase K solution (20 mg/mL). The mixture was incubated at 60° C.for 20 min then 20 μL of the mixture was injected to IEX-HPLC andanalyzed.

Incubation Control Sample:

Mixture of 5 μL of 250 μM of each DNA solutions and 103 μL of 1×PBSbuffer were prepared and 20 μL of the mixture was analyzed by IEX-HPLCas controls in order to check the absolute quantification.

Example Analytical Method:

IEx-HPLC A: 10 mM TrisHCl, 50% ACN (pH 8.0) B: 10 mM TrisHCl, 800 mMNaCl, 50% ACN (pH 8.0)

C: Water-ACN (1:1, v/v)

Temp: 60° C. Column: DIONEX DNAPac PA-100, 250×4 mm Gradient:

Time Flow % A % B % C % D Curve 1 0.00 1.00 95.0 5.0 0.0 0.0 6 2 1.001.00 95.0 5.0 0.0 0.0 1 3 2.00 1.00 75.0 25.0 0.0 0.0 6 4 10.00 1.00 5.095.0 0.0 0.0 6 5 10.10 1.00 95.0 5.0 0.0 0.0 6 6 12.50 1.00 95.0 5.0 0.00.0 1

Washing:

Time Flow % A % B % C % D Curve 1 0.01 1.00 0.0 0.0 100.0 0.0 6 2 5.501.00 0.0 0.0 100.0 0.0 1 3 5.60 1.00 0.0 100.0 0.0 0.0 6 4 7.50 1.00 0.0100.0 0.0 0.0 1 5 7.60 1.00 95.0 5.0 0.0 0.0 6 6 12.50 1.00 95.0 5.0 0.00.0 1

Column Temperature: 60° C.

Washing was performed every after the sample run.Percentage of remained PS DNA was calculated by the analysis of theratio from the 0 h to 48 h using the area of integration of HPLCchromatogram.

Example 14. Example Analytical Results (FIG. 19)

Peak Assignments for FIG. 19 (Top Panel, M12-Exp11 B10, ONT-354, 30 Min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 2.341100.6 733.7 11.91 1390.6 1042.6 13.07 1500.08 1125.5 750.73 1805.291354.19 13.58 1603.39 1202.2 961.35 801.15 14.80 1589.9 1271.4 1059.518.59 1653.3 1323.3 1101.6 Retention Assignment based on mass match timeObserved 5′-p-RNA 3′-OH and 5′-OH, (minutes) MW fragment RNA DNA 2.342203.2  7mer 11.91 4176 13mer 13.07 4505.7 14mer 5418.87 17mer 13.584812.8 15mer 14.80 6362.5 20mer, ONT-387 18.59 6615.4 ONT-354

Peak Assignments for FIG. 19 (Bottom Panel, M12-Exp11 A10, ONT-315, 30Min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 4.011425.33 950.15 4.4 1100.83 733.69 4.94 1578.34 1051.54 6.21 1741.911161.89 870.37 1445.42 963.31 722.97 8.48 1610 1073.3 9.15 1391.2 1043.19.93 1763.4 1174.7 11.8 1602.3 1201.7 14.82 20.73 1809.94 1447.82 1205.9Retention Assignment based on mass match time Observed 5′-p-RNA 3′-OHand 5′-OH, (minutes) MW fragment RNA DNA 4.01 2853.45  9mer 4.4 2203.66 7mer 4.94 3158.47 10mer 6.21 3487.52 11mer 2892.84  9mer 8.48 3220.9410mer 9.15 4177 13mer 9.93 3528.88 11mer 11.8 4810 15mer 14.82 20mer,ONT-387 20.73 7244.3 ONT-315

Example 15. Example Analytical Results (FIG. 30)

Peak Assignments for FIG. 30 (Top Panel, M12-Exp11 D2, ONT-367, 30 Min)

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 2.361120.28 746.25 3.15 1292.41 861.32 4.04 975.92 4.49 1140.6 759.78 5.831305.21 869.65 652.31 6.88 1923.23 1281.69 961.28 9.32 1390.76 1043.29833.72 9.96 1783.85 1187.98 891.6 712.94 11.01 1936.14 1289.93 1501.521125.4 899.89 11.93 1405.25 1053.78 842.84 13.15 1514.72 1135.72 14.811609.95 1287.53 1072.58 18.33 1587.9 1270.2 1058.3 Retention Assignmentbased on mass match time Observed 5′-p-RNA 3′-OH and 5′-OH, (minutes) MWfragment RNA DNA 2.36 2242.56  7mer 3.15 2586.82  8mer 4.04 1953.84 6mer 4.49 2283.2  7mer 5.83 2612.42  8mer 6.88 3849.14 12mer 9.324175.28 13mer 9.96 3569.7 11mer 11.01 3874.28 12mer 4507.56 14mer 11.934218.75 13mer 13.15 4547.16 14mer 14.81 6441.8 20mer, ONT-388 18.336355.6 ONT-367

Peak Assignments for FIG. 30 (Bottom Panel, M12-Exp21 NM Plate1 (Pool)F11 ONT-406 30 Min

Retention time (minutes) (M-2)²⁻ (M-3)³⁻ (M-4)⁴⁻ (M-5)⁵⁻ (M-6)⁶⁻ 4.721140.6 759.78 9.46 1390.76 1043.29 833.72 16.45 1609.95 1287.53 1072.5819.48 1588.1 1270.4 1058.4 Retention Assignment based on mass match timeObserved 5′-p-RNA 3′-OH and 5′-OH, (minutes) MW fragment RNA DNA 4.722203.2 2283.2 7mer 9.46 4176 4175.28 13mer 16.45 6362.5 6441.8 20mer,ONT-388 19.48 6615.4 6355.9

Example 16. Example Preparation of Linkers

In some embodiments, the SP linker was prepared following the schemebelow:

Example 17. Example Designs of Base Sequence

As described in the present disclosure, the present disclosurerecognizes the importance of base sequence, e.g., for provided chirallycontrolled oligonucleotide composition. In some embodiments, the presentdisclosure, as exemplified herein, provides methods for designing basesequence for oligonucleotides, such as antisense oligonucleotides.

In some embodiments, among other things, bioinformatics is used todesign a sequence for a target, e.g., a disease-associated mutant alleleof Huntington's disease. The present example describes example stepsthat may be used for design antisense oligonucleotides for, e.g.,rs362268, rs362306, rs2530595, rs362331, rs362307, etc. In someembodiments, a provided methods comprising a step of examining sequencefeatures for off-target, binding affinity with target, contiguous Gs,and paliandromic moieties. In some embodiments, a provided methodscomprising a step of examining off-target effects in the presence ofmismatches. In some embodiments, a sequence found in a target comprisinga characteristic sequence element, e.g., a mutation, a SNP, etc., andhaving a length of about 10-1000, e.g., about 10, about 20, about 30,about 40, about 50, about 60, about 70, about 80, about 90, about 100,about 110, about 120, about 130, about 140, about 150, about 200, about250, about 300, about 400, about 500, about 600, about 700, about 800,about 900, about 1000, about 2000, about 3000, about 4000, about 5000,etc., nucleotides are used in assays, e.g., RNase H assay, reporterassay, etc. In some embodiments, as in the present example, 40-bpflanking sequences for a SNP, e.g., rs362268, rs362306, rs2530595,rs362331, rs362307, etc., were used. A number of such sequences, forexample 6 to 12, could be readily assessed by provided methods. Exampletested sequences are listed in FIG. 42.

As described in the present disclosure and understood by a person havingordinary skill in the art, in some embodiments, assays, for example,RNase cleavage assay described herein, are useful in the assessment ofone or more features (e.g., rate, extent, and/or selectivity ofcleavage). In some embodiments, an RNase cleavage assay provides acleavage pattern of an oligonucleotide composition. In some embodiments,a composition of DNA oligonucleotides having the same sequence is used,an RNase H assay may provide a DNA cleavage pattern of the sequence. Insome embodiments, for generating a DNA cleavage pattern, all DNAoligonucleotides in the composition are identical. In some embodiments,when a stereorandom composition of all-phosphorothioate oligonucleotideshaving the same sequence is used, an RNase H assay may provide astereorandom cleavage pattern of the sequence. In some embodiments, forgenerating stereorandom cleavage pattern, all oligonucleotides in thestereorandom composition are identical. In some embodiments, when achirally controlled oligonucleotide composition is used, an RNase Hassay may provide a stereorandom cleavage pattern of the chirallycontrolled oligonucleotide composition. In some embodiments, forgenerating cleavage pattern of a chirally controlled oligonucleotidecomposition, all oligonucleotides in the chirally controlledoligonucleotide composition are identical. In some embodiments, an RNaseH assay provides cleavage rate information. In some embodiments, anRNase H assay provides relative cleavage extent, e.g., (cleavage at asite)/(all cleavage). In some embodiments, an RNase H assay providesabsolute cleavage extent, (cleaved target at a site)/(all target bothcleaved and non-cleaved). In some embodiments, an RNase H assay providesselectivity. In some embodiments, an RNase H assay provides suppressionlevel information.

In some embodiments, as exemplified herein, an RNase H assay providescleavage rates. For example results, see FIG. 31. P represents positionof mismatch in oligonucleotides from the 5′-end.

Analysis of human RNase H1 cleavage of a 25-mer RNA when hybridized withdifferent phosphorothioate oligonucleotides targeting rs362307 SNP wasperformed. WV-944 and WV-945 are 25mer RNAs which include WT and mutantvariant of rs362307, respectively. WV-936 to WV-941 are stereopure DNAswhile WV-904 to WV-909 are all stereorandom DNAs. All duplexes wereincubated with RNase H1C in the presence of 1×RNase H buffer at 37° C.Reactions were quenched at fixed time points by 30 mM Na₂EDTA. One tenthof this reaction mixture was injected on Reverse Phase HPLC and peakareas were measured for full length RNA remaining in the reactionmixtures at different time points. Cleavage rates were determined byplotting these peak areas with respective time points. In someembodiments, differentiation between rates of cleavage of WT RNA vs. muRNA was observed.

In some embodiments, as shown in FIG. 31, when position 11, 12 or 13 ofa sequence as counted from its 5′-terminus aligns with a SNP, orposition 8, 9 or 10 of a sequence as counted from its 3′-terminus alignswith a SNP, better cleavage selectivity was observed.

Example 18. Example Wing, Core, Wing-Core, Core-Wing and Wing-Core-WingDesigns

Among other things, the present disclosure provides various embodimentsof wing, core, wing-core, core-wing, and wing-core-wing structures. Insome embodiments, it was surprisingly found that oligonucleotides withwings comprising phosphate linkages and cores comprisingphosphorothioate linkages provided unexpectedly increased cleavageefficiency and selectivity. For example, see FIG. 32 C, F, G, H, etc.

Analysis of human RNase H1 cleavage of a 25-mer RNA when hybridized withdifferent chirally controlled oligonucleotide compositions targetingrs362307 SNP. WV-944 and WV-945 are 25mer RNAs which include WT andmutant variant of rs362307, respectively. WV-1085 to WV-1092 are allstereopure 2′-OMe/DNAs with mixed PO/PS backbone. All duplexes wereincubated with RNase H1C in the presence of 1×RNase H buffer at 37° C.Reactions were quenched at fixed time points by 30 mM Na₂EDTA. One tenthof this reaction mixture was injected on Reverse Phase HPLC and peakareas were measured for full length RNA remaining in the reactionmixtures at different time points. Cleavage rates were determined byplotting these peak areas with respective time points.

In some embodiments, 2′-OMe phosphate wings change cleavage rate and/orselectivity. In some embodiments, 2′-OMe phosphate wings change cleavagerate and selectivity. In some embodiments, 2′-OMe phosphate wings changecleavage rate or selectivity. In some embodiments, 2′-OMe phosphate wingchange cleavage rate. In some embodiments, 2′-OMe phosphate wing changecleavage rates of both the mutant and the wild-type allele. In someembodiments, 2′-OMe phosphate wing change cleavage selectivity. In someembodiments, 2′-OMe phosphate wing change cleavage pattern.

In some embodiments, incorporation of a phosphate internucleotidiclinkage surprisingly improves cleavage rate and/or selectivity. In someembodiments, incorporation of a phosphate internucleotidic linkagesurprisingly improves cleavage rate and selectivity. In someembodiments, incorporation of a phosphate internucleotidic linkagesurprisingly improves cleavage rate or selectivity. In some embodiments,incorporation of a phosphate internucleotidic linkage surprisinglyimproves cleavage rate. In some embodiments, a phosphateinternucleotidic linkage improves cleavage rates of both the mutant andthe wild-type allele, but at a greater level for the mutant than for thewild-type allele. In some embodiments, incorporation of a phosphateinternucleotidic linkage surprisingly improves cleavage selectivity.

In some embodiments, as demonstrated by data exemplified herein,stereopure oligonucleotide compositions provided surprisingly highcleavage rate and/or selectivity compared to corresponding stereorandomcompositions; for examples, see stereopure WV-1497/stereorandom WV-1092,905/937, 931/1087, etc.

Example 19. Example Cleavage Maps

As described herein, in some embodiments, an assay, such as RNase Hassay, provides cleavage maps for stereorandom or chirally controlledoligonucleotide compositions. Example cleavage maps are illustrated inFIG. 33, which exemplifies stereorandom cleavage patterns of multiplebase sequences. Additional cleavage maps are presented in FIG. 35, whichexemplifies, among other things, stereorandom cleavage patterns of basesequence having no nucleoside modifications (WV-905), as well as basesequence having nucleoside modifications.

Example cleavage patterns of chirally controlled stereopureoligonucleotide compositions are presented in FIG. 34. As described inthe present disclosure, major cleavage sites may be identified throughassays such RNase H assay from cleavage patterns. For example, forWV-937, the relative major cleavage site, as assessed by (cleavage atthe site/total cleavage) and reflected by the lengths of the arrows, arebetween GCGC and CCUU for the wild-type (2 internucleotidic linkagesaway from the SNP), and between CUGU and GCCC for the mutant (at the SNPsite, 0 internucleotidic linkage away from the SNP). In someembodiments, a relative major cleavage site is not necessarily anabsolute major cleavage site, which requires certain percentage of thetotal target, in this case, RNA, is cleaved at the site. For example, insome embodiments, the site between GCGC and CCUU for WV-937/wild type isnot a major cleavage site if over 20% of total target needs to becleaved at a site for a site to qualify as a major cleavage site (seeFIG. 32, M); the site between CUGU and GCCC for WV-937/mutant remains amajor cleavage site if the threshold for a major site is 20% of totaltarget being cleaved at that site.

In some embodiments, different oligonucleotide compositions havedifferent cleave rates. In some embodiments, cleavage maps are generatedat different time points. For example, for an oligonucleotidecomposition having a faster cleavage rate, its cleavage map can begenerated at an earlier time point (e.g., 5 minutes, 10 minutes, 15minutes, etc.) than an oligonucleotide composition having a slowercleavage rate (e.g., 30 minutes, 45 minutes, 60 minutes, etc.).

In some embodiments, when cleavage products at only one site can beidentified by an analytical methods, e.g., HPLC, HPLC-MS, etc., thecorresponding cleavage pattern is considered to have a single cleavagesite. In some embodiments, when greater than about 90%, greater thanabout 91%, greater than about 92%, greater than about 93%, greater thanabout 94%, greater than about 95%, greater than about 96%, greater thanabout 97%, greater than about 98%, greater than about 99%, or greaterthan about 99.5% of total cleavage occurs at a site, the correspondingcleavage pattern may be considered to have a single cleavage site. Insome embodiments, as understood by a person having ordinary skill in theart, selectivity in e.g., cells, tissues, organs, subjects, etc., may behigher than that observed in an RNase H assay. In some embodiments, asite having greater than about 90%, greater than about 91%, greater thanabout 92%, greater than about 93%, greater than about 94%, greater thanabout 95%, greater than about 96%, greater than about 97%, greater thanabout 98%, greater than about 99%, or greater than about 99.5% of totalcleavage in an RNase H assay may have higher selectivity in cells,tissues, organs, or subjects. In some embodiments, a site having greaterthan about 90% of total cleavage in an RNase H assay may be the onlycleavage site (e.g., greater than about 99%, greater than about 99.5%,100%, etc.) in cells, tissues, organs, or subjects.

In some embodiments, selectivity may be assessed by comparing absolutevalues of remaining transcripts (or representative sequences thereof,such as RNA sequences used in examples described herein) of a targetsequence and a similar sequence, e.g., RNA, or representative syntheticsequences, of a mutant allele as a target, and a wild-type allele as asimilar sequence. In some embodiments, selectivity may be assessed bycomparing absolute amounts of remaining transcripts (or representativesequences thereof, such as RNA sequences used in examples describedherein) of a target sequence and a similar sequence, when the startingamounts are the same. In some embodiments, selectivity may be assessedby percentages of cleavage of transcripts (or representative sequencesthereof, such as RNA sequences used in examples described herein) of atarget sequence and a similar sequence. In some embodiments, selectivitymay be assessed by comparing ratios of cleaved and non-cleavedtranscripts (or representative sequences thereof, such as RNA sequencesused in examples described herein).

In some embodiments, selectivity can be assessed by one or more assaysexemplified herein. In some embodiments, selectivity can be measured byan RNase H cleavage assay. For example, selective cleavage of a target(e.g., RNA from a mutant allele) can be measured by a biochemical RNaseH cleavage assay, wherein cleavage of a mutant target sequence iscompared to that of a wild-type RNA sequence, and the selectivity can berepresented by either the rate of cleavage, ratio of cleaved mutant RNAand wild-type RNA at a time point, or ratio of remaining mutant RNA andwild-type RNA at a time point, or combinations thereof. In someembodiments, a time point is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60 or more minutes. In some embodiments, a time point is 10 minutes. Insome embodiments, a time point is 15 minutes. In some embodiments, atime point is 20 minutes. In some embodiments, a time point is 25minutes. In some embodiments, a time point is 30 minutes. In someembodiments, a time point is 35 minutes. In some embodiments, a timepoint is 40 minutes. In some embodiments, a time point is 45 minutes. Insome embodiments, a time point is 50 minutes. In some embodiments, atime point is 55 minutes. In some embodiments, a time point is 60minutes. In some embodiments, a time point is 60 or minutes. A personhaving ordinary skill in the art understands how to choose a time point,for example, for cleavage shown in FIG. 32, one or more time points of5, 10, 15, 20, 20, 45 and 60 minutes can be chosen to assessselectivity. In some embodiments, selectivity can be measured by ratiosof IC₅₀ for target (e.g., mutant) and non-target (e.g, wild-type)sequences, e.g., from cell-based assays or animal models.

Example HPLC-MS traces are presented in FIG. 36. In some embodiments,example RNase H assay conditions were described below.

DNA/RNA Duplex Preparation: Oligonucleotide concentrations weredetermined by measuring the absorbance in water at 260 nm. DNA/RNAduplexes were prepared by mixing equimolar solutions oligonucleotideswith each strand concentration of 20 μM. The mixtures were heated at 90°C. for 2 minutes in water bath and were cooled down slowly over severalhours.

Human RNase H Protein Expression and Purification: Human RNase HC clonewas obtained from Prof. Wei Yang's laboratory at NIH Bethesda. Theprotocol for obtaining this human RNase HC (residues 136-286) has beendescribed (Nowotny, M. et al. Structure of Human RNase H1 Complexed withan RNA/DNA Hybrid: Insight into HIV Reverse Transcription. MolecularCell 28, 264-276, (2007)). The protein expression was carried out byfollowing reported protocol with the exception that the resultingprotein had an N-terminal His6 tag (SEQ ID NO: 1552). BL21(DE3) E. colicells in LB medium were used for protein expression. Cells were grown at37° C. till OD_(600nm) reached around 0.7. The cultures were then cooledand 0.4 mM IPTG was added to induce protein expression overnight at 16°C. E. coli extract was prepared by sonication in buffer A (40 mM NaH₂PO₄(pH 7.0), 1 M NaCl, 5% glycerol, 2.8 mM β-mercaptoethanol and 10 mMimidazole) with the addition of protease inhibitors (Sigma-Aldrich). Theextract was purified by Ni affinity column using buffer A plus 60 mMimidazole. The protein was eluted with a linear gradient of 60 to 300 mMimidazole. The protein peak was collected and was further purified on aMono S column (GE Healthcare) with a 100 mM-500 mM gradient of NaCl inbuffer B. Fractions containing RNase HC were concentrated to 0.3 mg/mLin the storage buffer (20 mM HEPES (pH 7.0), 100 mM NaCl, 5% glycerol,0.5 mM EDTA, 2 mM DTT) and stored at −20° C. 0.3 mg/mL enzymeconcentration corresponds to 17.4 μM based on its reported extinctioncoefficient (32095 cm⁻¹ M⁻¹) and MW (18963.3 Da units).

In a 96-well plate, to 50 μL DNA/RNA duplex (20 μM) was added 10 μL of10×RNase H buffer followed by 30 μL water. The mixture was incubated at37° C. for a few minutes and then 10 μL of 0.2 μM stock solution ofenzyme was added to give a total volume of 100 μL with finalsubstrate/enzyme concentration 10 μM/0.02 μM (500:1) and was furtherincubated at 37° C. Various ratios of the DNA/RNA duplex to RNase Hprotein were previously studied using these conditions to find thisoptimal ratio (500:1) to study the kinetics. The reactions were quenchedat different time points using 7 μL of 500 mM EDTA disodium solution inwater. For zero min time point, EDTA was added to the reaction mixturebefore the addition of enzyme. Controls were run to ensure that EDTA wasable to inhibit the enzyme activity completely. After all the reactionswere quenched 10 μL or 20 μL of each reaction mixture was injected on toLCMS-TOF using analytical column (Agilent Poroshell 120 EC-C18 2.7micron, 2.1×150 mm, Part #699775-902). Ratio of peak area of remainingfull length RNA to DNA in each reaction mixture was normalized againstthis ratio at zero point reaction to obtain the % of full length RNAremaining.

In some embodiments, an example HPLC condition is:

Eluant A=8 mM TEA, 200 mM HFIP in Water Eluant B=50:50 (Eluant A:Methanol) Column Temperature=50° C.

Auto sampler temperature=4° C.UV was recorded at 254 nm and 280 nm

LC Gradient Method

Time (min) Flow (mL/min) % A % B 1 0.0 0.2 90 10 2 15.0 0.2 65 35 3 22.00.2 40 60 4 25.0 0.2 5.0 95.0 5 25.5 0.2 90 10 6 30 0.2 90 10

Example 20. Example Assays for Assessing Oligonucleotides

In some embodiments, the present disclosure provides reporter assays forassessing properties of provided oligonucleotides and compositions. Insome embodiments, a provided reporter assay is a dual-luciferase assay,e.g., as described below.

Determination of mRNA inhibition by oligonucleotides using Dual GloLuciferase System: The psiCheck2 vector system from Promega is acommercially available vector which encodes both Photinus pyralis andRenilla Reniformis luciferase genes on a single plasmid with a multiplecloning site in the 3′ UTR of Renilla luciferase for insertion ofoligonucleotides encoding the miRNA target sites (or other clonedregulatory sequences, such as target 3′UTRs). A 250 base pairs fragmentcontaining the targeting region of interest and its reverse complementand having appropriate overhanging bases corresponding to therestriction enzyme(s) used to digest the psiCheck vector was cloned intothe psiCHECK-2 vector (Promega, C8021) between NotI and XhoI restrictionenzyme sites. The vector containing the insert was sequenced to confirmcorrect orientation of insert, expanded and purified. Multiple vectorswere generated using above design for SNPs of interest. In a typicalco-transfection experiment, after the cells were at the correct density(30 to 40% confluency), oligonucleotides and vector werereverse-transfected using Lipofectamine 2000 (Life Technologies).Effects of oligonucleotides on target mRNA can be seen as early as 24 hpost-transfection and were still present after 48 h. 24 hour or 48 hoursafter transfection of psiCheck vectors, cells were assayed forluciferase activity. Briefly, cells are washed with PBS, lysed inpassive lysis buffer, luciferin reagents were added, and samples wereread on a Spectramax M5 instrument (Molecular Devices). Measurementswere taken at vector concentrations of 20 ng per well of a 96-wellplate. The experiments were performed at various oligonucleotideconcentrations (30, 10 and 3.3 nM) and two time points (24 and 48hours). The relative levels of Renilla luciferase vs. Firefly weremeasured for untreated cells and cells which were treated witholigonucleotides targeting Renilla (WV-975) to measure the maximumRenilla knockdown. Control oligonucleotides (e.g., WV-437, WV-993, etc.)were chosen to normalize the R/F levels. In some embodiments, dualluciferase reporter assay was used to assess oligonucleotides in Cos7cell line. In some embodiments, the cell line was cotransfected for 24hrs with oligonucleotides and either of the psiCHECK2 plasmids,including rs362307 (T) or rs362307 (C) SNP. In some embodiments,rs362307 (T) and rs362307 (C) are referred as mu and wt.

Various chirally controlled and stereorandom oligonucleotidecompositions were tested at 30 nM using the dual-luciferase assay. Foroligonucleotides with mismatch at the same position (e.g., positions 8,9, 10, 11, 12 and 13 relative to the 5′-end), chirally controlledcompositions maintained high levels of wide-type measurements.

In some embodiments, when tested at 30 nM, WV-1092 selectivelysuppressed expression of the mutant sequence at 24 and/or 48 hours asshown by the dual luciferase reporter assay. In some embodiments, theobserved selectivity for WV-1092 at 30 nM 24 hours and/or 48 hours wasseveral fold more than other oligonucleotide compositions, e.g., WV-917,WV-1497, certain P12 stereopure oligonucleotides, etc. In someembodiments, at 30 nM, 48 hours, WV-1092 maintained over 90% wild-type,and decreased the mutant to about 30%, while WV-917 decreased thewild-type to about 60%, and mutant to about 30%. Oligonucleotides aretested at multiple conditions (e.g., concentrations, time points, etc.),and show improved properties, e.g., activity, selectivity, etc.

As understood by a person having ordinary skill in the art,oligonucleotide properties, e.g., activity, selectivity, etc., may beassessed by many other assays, such as cell-based assays, animal models,etc. In some embodiments, allele-specific suppression may be tested incells and animal models using similar procedures as described in Hohjoh,Pharmaceuticals 2013, 6, 522-535; US patent application publication US2013/0197061; Østergaard et al., Nucleic Acids Research 2013, 41(21),9634-9650; Jiang et al., Science 2013, 342, 111-114; and U.S. Pat. No.9,006,198. In some embodiments, selectivity can be assessed by IC₅₀values for the wild-type and the mutant allele. Provided compositions,including those targeting SNPs associated with Huntington's disease,suppress disease-associated alleles selectively over wild type alleles.

Example 21. Example Methods for Preparing Oligonucleotides andCompositions Abbreviations

AMA: conc. NH₃—40% MeNH₂ in H₂O (1:1, v/v)CMIMT: N-cyanomethylimidazolium triflateDBU: 1,8-diazabicyclo[5.4.0]undec-7-eneDCA: dichloroacetic acidDCM: dichloromethane, CH₂Cl₂DMTr: 4,4′-dimethoxytritylDVB: divinylbenzeneHCP: highly cross-linked polystyrene (contains 50% DVB, non-swellingpolystyrene)

MeIm: N-methylimidazole

MQ: water obtained from “Milli-Q Reference”PhIMT: N-phenylimidazolium triflatePOS: 3-phenyl-1,2,4-dithiazolin-5-onePS200: primer support 200, commercially available from GE HealthcarePS5G: primer support 5G, commercially available from GE HealthcareTBAF: tetrabutylammonium fluorideTBHP: tert-butylhydroperoxideTEAA: triethylammonium aceate

Solid support: Various types of solid support (varied nucleosidesloading) were tested. In some embodiments, HCP>PS5G≈PS200≥CPG. In someembodiments, a solid support is HCP. In some embodiments, a solidsupport is PS5G. In some embodiments, a solid support is PS200. In someembodiments, a solid support is CPG. For nucleosides loading, variousrange (30-300 μmol/g) were tested. In some embodiments, 70-80 μmol/gloading performed than others. In some embodiments, nucleoside loadingis 70-80 μmol/g. CPG was purchased from various suppliers (GlenReseach,LinkTechnologies, ChemGenes, PrimeSynthesis, and 3-Prime).

Various linkers were tested and can be used. In some embodiments, duringpreparation of chirally controlled oligonucleotide compositions by usingDPSE-type chemistry, SP-linker was used.

Various activators were prepared and/or purchased, and evaluated. Insome embodiments, for DPSE-type chemistry, CMIMT was used.

Example Analytical conditions:

1) RP-UPLC-MS

-   -   System: Waters, Acquity UPLC I-Class, Xevo G2-Tof    -   Column: Waters, BEH C18, 1.7 m, 2.1×150 mm    -   Temp. & Flow rate: 55° C., 0.3 mL/min    -   Buffer: A: 0.1M TEAA; B: MeCN    -   Gradient: % B: 1-30%/30 min

2) AEX-HPLC

-   -   System: Waters, Alliance e2695    -   Column: Thermo, DNAPac PA-200, 4×250 mm    -   Temp. & Flow rate: 50° C., 1 mL/min    -   Buffer: A: 20 mM NaOH; B: A+1M NaClO₄    -   Gradient: % B: 10-50%/30 min

Example procedure for the synthesis of chrial-oligos (1 μmol scale):

Automated solid-phase synthesis of chiral-oligos was performed accordingto example cycles shown herein. After the synthesis cycles, the resinwas treated with 0.1M TBAF in MeCN (1 mL) for 2 h (30 min usuallyenough) at room temperature, washed with MeCN, dried, and add AMA (1 mL)for 30 min at 45° C. The mixture was cooled to room temperature and theresin was removed by membrane filtration. The filtrate was concentratedunder reduced pressure to about 1 mL. The residue was diluted with 1 mLof H₂O and analyzed by AEX-HPLC and RP-UPLC-MS (example conditions:refer to the analytical conditions).

waiting step operation reagents and solvent volume time 1 detritylation3% DCA in toluene 10 mL 65 s 2 coupling 0.15M monomer in ^(i)PrCN + 0.5mL 5 min 0.5M CMIMT in MeCN 3 capping 20% Ac₂O, 30% 2,6- 1.2 mL 60 slutidine in MeCN + 20% MeIm in MeCN 4 oxidation or 1.1M TBHP in DCM- 1.0mL 300 s sulfurization decane or 0.1M POS in MeCN

As described, TBAF treatment can provide better results, for example,less desulfurization. In some embodiments, SP linker provided betteryields and/or purity through, without the intention to be limited bytheory, better stability during chiral auxiliary removal as described.In some embodiments, fluoro-containing reagents such as HF—NR₃ (e.g.,HF-TEA (triethylamine)), provided better yields and/or purity whensuccinyl linker was used by, without the intention to be limited bytheory, less cleavage during chiral auxiliary removal. In someembodiments, after synthesis, the resin was treated with 1M TEA-HF inDMF-H₂O (3:1, v/v; 1 mL) for 2 h at 50° C. PS5G support was washed withMeCN, H₂O, and add AMA (conc. NH₃—40% MeNH₂ (1:1, v/v)) (1 mL) for 45min at 50° C. The mixture was cooled to room temperature and the resinwas removed by membrane filtration (washed with H₂O for 2 mL). Thefiltrate was concentrated under reduced pressure until it becomes about1 mL. The residue was diluted with 1 mL of H₂O and analyzed by AEX-HPLCand RP-UPLC-MS (conditions: refer to the analytical conditions section).

Example procedure for the purification of chrial-oligos (1 μmol scale):in some embodiments, crude oligos were purified by AEX-MPLC according tothe following example conditions:

-   -   System: AKTA Purifier-10    -   Column: TOHSOH, DNA STAT, 4.6×100 mm    -   Temp. & Flow rate: 60° C., 0.5 mL/min    -   Buffer: A: 20 mM Tris-HCl (pH 9.0)+20% MeCN, B: A+1.5M NaCl    -   Gradient: % B: 20-70%/25 CV (2%/CV)

All fractions were analyzed by analytical AEX-HPLC, and fractionscontaining chiral oligo more than 80% purity were corrected and desaltedby Sep-Pak Plus tC18 (WAT036800) using example conditions below:

1. Conditioning Sep-Pak Plus with 15 mL of MeCN.

2. Rinse cartridge with 15 mL of 50% MeCN/MQ.

3. Equilibrate cartridge with 30 mL of MQ.

4. Load sample, and wash with 40 mL of MQ.

5. Elute chiral oligos with 10 mL of 50% MeCN/MQ.

Eluted sample were evaporated under reduced pressure to remove MeCN, andlyophilized. The product were dissolved in MQ (1 mL), filtered by 0.2 μmmesh syringe filter, and analyzed. After yield calculation by UVabsorbance, the preparation was lyophilized again.

Example methods, conditions and reagents were described in, e.g., JP2002-33436, WO2005/092909, WO2010/064146, WO2012/039448, WO2011/108682,WO2014/010250, WO2014/010780, WO2014/012081, etc., and may be useful forpreparing provided oligonucleotides and/or compositions.

Additional example oligonucleotides are listed below. In someembodiments, one or more of the oligonucleotides below are used ascontrols. In some embodiments, one or more of the oligonucleotides beloware RNA sequences as cleavage targets in one or more assays.

TABLE 5N Example Control Oligonucleotides. WV-975G*T*A*G*G*A*G*T*A*G*T*G*A*A*A*G*G*C*C*A (SEQ ID NO: 783) WV-1061mG*mU*mA*mG*mG*A*G*T*A*G*T*G*A*A*A*mG*mG*mC *mC*mA (SEQ ID NO: 784)WV-1062 mGmUmAmGmG*A*G*T*A*G*T*G*A*A*A*mGmGmCmCmA (SEQ ID NO: 785)WV-1063 mG*mU*mA*mG*mG*A*G*T*A*G*T*G*A*A*A*G*G*C*C* A (SEQ ID NO: 786)WV-1064 mC*mU*mC*mU*mU*A*C*T*G*T*G*C*T*G*T*mG*mG*mA*mC*mA (SEQ ID NO: 787) WV-1065mCmUmCmUmU*A*C*T*G*T*G*C*T*G*T*mGmGmAmCmA (SEQ ID NO: 788) WV-1066mC*mU*mC*mU*mU*A*C*T*G*T*G*C*T*G*T*G*G*A*C*A (SEQ ID NO: 789) WV-993mC*mC*mU*mU*mC*C*C*T*G*A*A*G*G*T*T*mC*mC*mU *mC*mC (SEQ ID NO: 790)WV-975 All DNA, Stereorandom PS, positive control for Renilla luciferasein psiCHECK2 plasmid WV-10615-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, +ve Luciferasecontrol for psiCHECK2 WV-10625-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings:+ve Luciferase control for psiCHECK2 WV-10635-15 (2′-OMe-DNA), stereorandom PS, +ve Luciferase control for psiCHECK2WV-1064 5-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, NegativeLuciferase control for psiCHECK2 WV-10655-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, PO in the wings:Negative Luciferase control for psiCHECK2 WV-10665-15 (2′-OMe-DNA), stereorandom PS, Negative Luciferase controlfor psiCHECK2 WV-9935-10-5 (2′-OMe-DNA-2′-OMe), stereorandom PS, NegativeLuciferase control for psiCHECK2

TABLE 6N Example RNA Sequences. WV-944rUrUrUrGrGrArArGrUrCrUrGrCrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 791)WV-945 rUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC(SEQ ID NO: 792) WV-1073rGrArGrCrCrUrUrUrGrGrArArGrUrCrUrGrCrGrCrCrCrUrUrGrUrGrCrCrCrUrGrCrCrU (SEQ ID NO: 793) WV-1074rGrArGrCrCrUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrCrUrGrCrCrU (SEQ ID NO: 794) WV-950rGrGrUrUrGrUrUrGrCrCrArGrGrUrUrArCrArGrCrUrGrCrUrC (SEQ ID NO: 795)WV-951 rGrGrUrUrGrUrUrGrCrCrArGrGrUrUrGrCrArGrCrUrGrCrUrC(SEQ ID NO: 796) WV-958rCrCrUrCrCrUrGrCrArGrGrCrUrGrGrGrUrGrUrUrGrGrCrCrC (SEQ ID NO: 797)WV-959 rCrCrUrCrCrUrGrCrArGrGrCrUrGrGrCrUrGrUrUrGrGrCrCrC(SEQ ID NO: 798) ONT-453rGrGrUrGrArUrGrArCrArArUrUrUrArUrUrArArU (SEQ ID NO: 799) ONT-454rGrGrUrGrArUrGrGrCrArArUrUrUrArUrUrArArU (SEQ ID NO: 800) WV-944rs362307 WT WV-945 rs362307 mu WV-1073 rs362307 WT WV-1074 rs362307 muWV-950 rs362306 WT WV-951 rs362306 mu WV-958 rs362268 WT WV-959 rs362268mu ONT-453 rs7685686 WT ONT-454 rs7685686 mu

Example 22. Example Oligonucleotides

Additional example oligonucleotides are listed below in Table 8.

TABLE 8 HTT Oligonucleotides. SEQ SEQ SEQ Naked ID ID ID Sequence NO:Modified Sequence NO: Stereochemistry Comment 1 Comment 2 ONT- ATTAATA801 A * T * T * A * A * T * A * A * 1153 XXXXXXXXX Stereorandom HttHtt SNP 450 AATTGTC A * T * T * G * T * C * A * T * XXXXXXXXX sequencers7685686 ATCACC C * A * C * C X ONT- ATTAATA 802A * ST * ST * SA * SA * ST * 1154 SSSSSSSSSSS Stereopure Htt Htt SNP 451AATTGTC SA * SA * SA * ST * ST * SG * SSRSSSSS sequence I rs7685686ATCACC ST * SC * RA * ST * SC * SA * SC * SC ONT- ATTAATA 803A * ST * ST * SA * SA * ST * 1155 SSSSSSSSSSS Stereopure Htt Htt SNP 452AATTGTC SA * SA * SA * ST * ST * SG * SSSRSSSS sequence II rs7685686ATCACC ST * SC * SA * RT * SC * SA * SC * SC ONT- GGUGAUG 804rGrGrUrGrArUrGrArCrArArUr 1156 OOOOOOOOO RNA against Htt Htt SNP 453ACAAUUU UrUrArUrUrArArU OOOOOOOOO sequence Mutant rs7685686 AUUAAU OONT- GGUGAUG 805 rGrGrUrGrArUrGrGrCrArArUr 1157 OOOOOOOOORNA against Htt Htt SNP 454 GCAAUUU UrUrArUrUrArArU OOOOOOOOOsequence Wild rs7685686 AUUAAU O Type WV- UUUGGAA 806rUrUrUrGrGrArArGrUrCrUrGr 1158 OOOOOOOOO wtRNA muHTT 902 GUCUGCGCrGrCrCrCrUrUrGrUrGrCrCrC OOOOOOOOO SNP CCCUUGU OOOOOO 362307 GCCC WV-UUUGGAA 807 rUrUrUrGrGrArArGrUrCrUrGr 1159 OOOOOOOOO mRNA muHTT 903GUCUGUG UrGrCrCrCrUrUrGrUrGrCrCrC OOOOOOOOO SNP CCCUUGU OOOOOO 362307GCCC WV- GGGCACA 808 G * G * G * C * A * C * A * A * 1160 XXXXXXXXXASO1 All DNA; muHTT 904 AGGGCAC G * G * G * C * A * C * A * G *XXXXXXXXX stereorandom PS SNP AGACTT A * C * T * T X 362307 WV- GGCACAA809 G * G * C * A * C * A * A * G * 1161 XXXXXXXXX ASO2 All DNA; muHTT905 GGGCACA G * G * C * A * C * A * G * A * XXXXXXXXX stereorandom PSSNP GACTTC C * T * T * C X 362307 WV- GCACAAG 810G * C * A * C * A * A * G * G * 1162 XXXXXXXXX ASO3 All DNA; muHTT 906GGCACAG G * C * A * C * A * G * A * C * XXXXXXXXX stereorandom PS SNPACTTCC T * T * C * C X 362307 WV- CACAAGG 811C * A * C * A * A * G * G * G * 1163 XXXXXXXXX ASO4 All DNA; muHTT 907GCACAGA C * A * C * A * G * A * C * T * XXXXXXXXX stereorandom PS SNPCTTCCA T * C * C * A X 362307 WV- ACAAGGG 812A * C * A * A * G * G * G * C * 1164 XXXXXXXXX ASO5 All DNA; muHTT 908CACAGAC A * C * A * G * A * C * T * T * XXXXXXXXX stereorandom PS SNPTTCCAA C * C * A * A X 362307 WV- CAAGGGC 813C * A * A * G * G * G * C * A * 1165 XXXXXXXXX ASO6 All DNA; muHTT 909ACAGACT C * A * G * A * C * T * T * C * XXXXXXXXX stereorandom PS SNPTCCAAA C * A * A * A X 362307 WV- GGGCACA 814 mG * mG * mG * mC * mA * C1166 XXXXXXXXX ASO7 5-15 (2′- muHTT 910 AGGGCAC* A * A * G * G * G * C * A * C XXXXXXXXX OMe-DNA); SNP AGACTT* A * G * A * C * T * T X stereorandom PS 362307 WV- GGCACAA 815mG * mG * mC * mA * mC * A 1167 XXXXXXXXX ASO8 5-15 (2′- muHTT 911GGGCACA * A * G * G * G * C * A * C * A XXXXXXXXX OMe-DNA); SNP GACTTC* G * A * C * T * T * C X stereorandom PS 362307 WV- GCACAAG 816mG * mC * mA * mC * mA * A 1168 XXXXXXXXX ASO9 5-15 (2′- muHTT 912GGCACAG * G * G * G * C * A * C * A * G XXXXXXXXX OMe-DNA); SNP ACTTCC* A * C * T * T * C * C X stereorandom PS 362307 WV- CACAAGG 817mC * mA * mC * mA * mA * G 1169 XXXXXXXXX ASO10 5-15 (2′- muHTT 913GCACAGA * G * G * C * A * C * A * G * A XXXXXXXXX OMe-DNA); SNP CTTCCA* C * T * T * C * C * A X stereorandom PS 362307 WV- ACAAGGG 818mA * mC * mA * mA * mG * G 1170 XXXXXXXXX ASO11 5-15 (2′- muHTT 914CACAGAC * G * C * A * C * A * G * A * C XXXXXXXXX OMe-DNA); SNP TTCCAA* T * T * C * C * A * A X stereorandom PS 362307 WV- CAAGGGC 819mC * mA * mA * mG * mG * G 1171 XXXXXXXXX ASO12 5-15 (2′- muHTT 915ACAGACT * C * A * C * A * G * A * C * T XXXXXXXXX OMe-DNA); SNP TCCAAA* T * C * C * A * A * A X stereorandom PS 362307 WV- GGGCACA 820mG * mG * mG * mC * mA * C 1172 XXXXXXXXX ASO13 5-10-5 (2′- muHTT 916AGGGCAC * A * A * G * G * G * C * A * C XXXXXXXXX OMe-DNA-2′- SNP AGACUU* A * mG * mA * mC * mU * X OMe); 362307 mU stereorandom PS WV- GGCACAA821 mG * mG * mC * mA * mC * A 1173 XXXXXXXXX ASO14 5-10-5 (2′- muHTT917 GGGCACA * A * G * G * G * C * A * C * A XXXXXXXXX OMe-DNA-2′- SNPGACUUC * G * mA * mC * mU * mU * X OMe); 362307 mC stereorandom PS WV-GCACAAG 822 mG * mC * mA * mC * mA * A 1174 XXXXXXXXX ASO15 5-10-5 (2′-muHTT 918 GGCACAG * G * G * G * C * A * C * A * G XXXXXXXXX OMe-DNA-2′-SNP ACUUCC * A * mC * mU * mU * mC * X OMe); 362307 mC stereorandom PSWV- CACAAGG 823 mC * mA * mC * mA * mA * G 1175 XXXXXXXXXASO16 5-10-5 (2′- muHTT 919 GCACAGA * G * G * C * A * C * A * G * AXXXXXXXXX OMe-DNA-2′- SNP CUUCCA * C * mU * mU * mC * mC * X OMe);362307 mA stereorandom PS WV- ACAAGGG 824 mA * mC * mA * mA * mG * G1176 XXXXXXXXX ASO17 5-10-5 (2′- muHTT 920 CACAGAC* G * C * A * C * A * G * A * C XXXXXXXXX OMe-DNA-2′- SNP TUCCAA* T * mU * mC * mC * mA * X OMe); 362307 mA stereorandom PS WV- CAAGGGC825 mC * mA * mA * mG * mG * G 1177 XXXXXXXXX ASO18 5-10-5 (2′- muHTT921 ACAGACT * C * A * C * A * G * A * C * T XXXXXXXXX OMe-DNA-2′- SNPTCCAAA * T * mC * mC * mA * mA * X OMe); 362307 mA stereorandom PS WV-GCACAAG 826 mG * mC * mA * mC * mA * 1178 XXXXXXXXX ASO19 8-7-5 (2′-muHTT 922 GGCACAG * mA * mG * mG * G * C * A * C XXXXXXXXX OMe-DNA-2′-SNP ACUUCC * A * G * A * mC * mU * mU * X OMe); 362307 mC * mCstereorandom PS WV- CACAAGG 827 mC * mA * mC * mA * mA * 1179 XXXXXXXXXASO20 7-7-6 (2′- muHTT 923 GCACAGA mG * mG * G * C * A * C * A *XXXXXXXXX OMe-DNA-2′- SNP CUUCCA G * A * mC * mU * mU * mC * X OMe);362307 mC * mA stereorandom PS WV- ACAAGGG 828 mA * mC * mA * mA * mG *1180 XXXXXXXXX ASO21 6-7-5 (2′- muHTT 924 CACAGACmG * G * C * A * C * A * G * A XXXXXXXXX OMe-DNA-2′- SNP UUCCAA* mC * mU * mU * mC * mC * X OMe); 362307 mA * mA stereorandom PS;PO in the wings WV- CAAGGGC 829 mC * mA * mA * mG * mG * G 1181XXXXXXXXX ASO22 5-7-8 (2′- muHTT 925 ACAGACU* C * A * C * A * G * A * mC * XXXXXXXXX OMe-DNA-2′- SNP UCCAAAmU * mU * mC * mC * mA * X OMe); 362307 mA * mA stereorandom PS;PO in the wings WV- GCACAAG 830 mGmCmAmCmAmAmGmG * 1182 OOOOOOOXXASO23 8-7-5 (2′- muHTT 926 GGCACAG G * C * A * C * A * G * A * XXXXXXOOOOMe-DNA-2′- SNP ACUUCC mCmUmUmCmC O OMe); 362307 stereorandom PS;PO in the wings WV- CACAAGG 831 mCmAmCmAmAmGmG * G * 1183 OOOOOOXXXASO24 7-7-6 (2′- muHTT 927 GCACAGA C * A * C * A * G * A * XXXXXOOOOOMe-DNA-2′- SNP CUUCCA mCmUmUmCmCmA O OMe); 362307 stereorandom PS;PO in the wings WV- ACAAGGG 832 mAmCmAmAmGmG * G * C * 1184 OOOOOXXXXASO25 6-7-5 (2′- muHTT 928 CACAGAC A * C * A * G * A * XXXXOOOOOOMe-DNA-2′- SNP UUCCAA mCmUmUmCmCmAmA O OMe); 362307 stereorandom PS;PO in the wings WV- CAAGGGC 833 mCmAmAmGmG * G * C * A * 1185 OOOOXXXXXASO26 5-7-8 (2′- muHTT 929 ACAGACU C * A * G * A * XXXOOOOOO OMe-DNA-2′-SNP UCCAAA mCmUmUmCmCmAmAmA O OMe); 362307 stereorandom PS;PO in the wings WV- GGGCACA 834 mGmGmGmCmA * C * A * A * 1186 OOOOXXXXXASO27 5-10-5 (2′- muHTT 930 AGGGCAC G * G * G * C * A * C * A *XXXXXXOOO OMe-DNA-2′- SNP AGACUU mGmAmCmUmU O OMe); 362307stereorandom PS; PO in the wings WV- GGCACAA 835mGmGmCmAmC * A * A * G * 1187 OOOOXXXXX ASO28 5-10-5 (2′- muHTT 931GGGCACA G * G * C * A * C * A * G * XXXXXXOOO OMe-DNA-2′- SNP GACUUCmAmCmUmUmC O OMe); 362307 stereorandom PS; PO in the wings WV- GCACAAG836 mGmCmAmCmA * A * G * G * 1188 OOOOXXXXX ASO29 5-10-5 (2′- muHTT 932GGCACAG G * C * A * C * A * G * A * XXXXXXOOO OMe-DNA-2′- SNP ACUUCCmCmUmUmCmC O OMe); 362307 stereorandom PS; PO in the wings WV- CACAAGG837 mCmAmCmAmA * G * G * G * 1189 OOOOXXXXX ASO30 5-10-5 (2′- muHTT 933GCACAGA C * A * C * A * G * A * C * XXXXXXOOO OMe-DNA-2′- SNP CUUCCAmUmUmCmCmA O OMe); 362307 stereorandom PS; PO in the wings WV- ACAAGGG838 mAmCmAmAmG * G * G * C * 1190 OOOOXXXXX ASO31 5-10-5 (2′- muHTT 934CACAGAC A * C * A * G * A * C * T * XXXXXXOOO OMe-DNA-2′- SNP TUCCAAmUmCmCmAmA O OMe); 362307 stereorandom PS; PO in the wings WV- CAAGGGC839 mCmAmAmGmG * G * C * A * 1191 OOOOXXXXX ASO32 5-10-5 (2′- muHTT 935ACAGACT C * A * G * A * C * T * T * XXXXXXOOO OMe-DNA-2′- SNP TCCAAAmCmCmAmAmA O OMe); 362307 stereorandom PS; PO in the wings WV- GGGCACA840 G * SG * SG * SC * SA * SC * 1192 SSSSSSSSSSS ASO33 Stereopure muHTT936 AGGGCAC SA * SA * SG * SG * SG * SC * SSRSSSSS DNA; One Rp; SNPAGACTT SA * SC * RA * SG * SA * SC * position 14 362307 ST * ST WV-GGCACAA 841 G * SG * SC * SA * SC * SA * 1193 SSSSSSSSSSSASO34 Stereopure muHTT 937 GGGCACA SA * SG * SG * SG * SC * SA *SRSSSSSS DNA; One Rp; SNP GACTTC SC * RA * SG * SA * SC * ST *position 13 362307 ST * SC WV- GCACAAG 842 G * SC * SA * SC * SA * SA *1194 SSSSSSSSSSS ASO35 Stereopure muHTT 938 GGCACAGSG * SG * SG * SC * SA * SC * RSSSSSSS DNA; One Rp; SNP ACTTCCRA * SG * SA * SC * ST * ST * position 12 362307 SC * SC WV- CACAAGG 843C * SA * SC * SA * SA * SG * 1195 SSSSSSSSSSR ASO36 Stereopure muHTT 939GCACAGA SG * SG * SC * SA * SC * RA * SSSSSSSS DNA; One Rp; SNP CTTCCASG * SA * SC * ST * ST * SC * position 11 362307 SC * SA WV- ACAAGGG 844A * SC * SA * SA * SG * SG * 1196 SSSSSSSSSRS ASO37 Stereopure muHTT 940CACAGAC SG * SC * SA * SC * RA * SG * SSSSSSSS DNA; One Rp; SNP TTCCAASA * SC * ST * ST * SC * SC * position 10 362307 SA * SA WV- CAAGGGC 845C * SA * SA * SG * SG * SG * 1197 SSSSSSSSRSS ASO38 Stereopure muHTT 941ACAGACT SC * SA * SC * RA * SG * SA * SSSSSSSS DNA; One Rp; SNP TCCAAASC * ST * ST * SC * SC * SA * position 9 362307 SA * SA WV- UUUGGAA 846rUrUrUrGrGrArArGrUrCrUrGr 1198 OOOOOOOOO HTT-rs362307 Huntington 944GUCUGCG CrGrCrCrCrUrUrGrUrGrCrCrC OOOOOOOOO human CCCUUGU OOOOOO GCCCWV- UUUGGAA 847 rUrUrUrGrGrArArGrUrCrUrGr 1199 OOOOOOOOO HTT-rs362307Huntington 945 GUCUGUG UrGrCrCrCrUrUrGrUrGrCrCrC OOOOOOOOO human CCCUUGUOOOOOO GCCC WV- GAGCAGC 848 G * A * G * C * A * G * C * T * 1200XXXXXXXXX HTT-rs362306 HTT- 948 TGCAACC G * C * A * A * C * C * T * G *XXXXXXXXX rs362306 TGGCAA G * C * A * A X WV- GGGCCAA 849G * G * G * C * C * A * A * C * 1201 XXXXXXXXX HTT-rs362268 HTT- 949CAGCCAG A * G * C * C * A * G * C * C * XXXXXXXXX rs362268 CCTGCAT * G * C * A X WV- GGUUGUU 850 rGrGrUrUrGrUrUrGrCrCrArGr 1202 OOOOOOOOOHTT- 950 GCCAGGU GrUrUrArCrArGrCrUrGrCrUrC OOOOOOOOO rs362306 UACAGCUOOOOOO GCUC WV- GGUUGUU 851 rGrGrUrUrGrUrUrGrCrCrArGr 1203 OOOOOOOOOHTT- 951 GCCAGGU GrUrUrGrCrArGrCrUrGrCrUrC OOOOOOOOO rs362306 UGCAGCUOOOOOO GCUC WV- GAGCAGC 852 G * SA * SG * SC * SA * SG * 1204SSSSSSSSSSR Stereopure PS HTT- 952 TGCAACC SC * ST * SG * SC * SA * RA *SSSSSSSS DNA; One Rp at rs362306 TGGCAA SC * SC * ST * SG * SG * SC *position 11 SA * SA WV- AGCAGCT 853 A * SG * SC * SA * SG * SC * 1205SSSSSSSSSRS Stereopure PS HTT- 953 GCAACCT ST * SG * SC * SA * RA * SC *SSSSSSSS DNA; One Rp at rs362306 GGCAAC SC * ST * SG * SG * SC * SA *position 10 SA * SC WV- GCAGCTG 854 G * SC * SA * SG * SC * ST * 1206SSSSSSSSRSS Stereopure PS HTT- 954 CAACCTG SG * SC * SA * RA * SC * SC *SSSSSSSS DNA; One Rp at rs362306 GCAACA ST * SG * SG * SC * SA * SA *position 9 SC * SA WV- CAGCTGC 855 C * SA * SG * SC * ST * SG * 1207SSSSSSSRSSS Stereopure PS HTT- 955 AACCTGG SC * SA * RA * SC * SC * ST *SSSSSSSS DNA; One Rp at rs362306 CAACAA SG * SG * SC * SA * SA * SC *position 8 SA * SA WV- AGCTGCA 856 A * SG * SC * ST * SG * SC * 1208SSSSSSRSSSS Stereopure PS HTT- 956 ACCTGGC SA * RA * SC * SC * ST * SG *SSSSSSSS DNA; One Rp at rs362306 AACAAC SG * SC * SA * SA * SC * SA *position 7 SA * SC WV- GCTGCAA 857 G * SC * ST * SG * SC * SA * 1209SSSSSRSSSSS Stereopure PS HTT- 957 CCTGGCA RA * SC * SC * ST * SG * SG *SSSSSSSS DNA; One Rp at rs362306 ACAACC SC * SA * SA * SC * SA * SA *position 6 SC * SC WV- CCUCCUG 858 rCrCrUrCrCrUrGrCrArGrGrCrU 1210OOOOOOOOO HTT- 958 CAGGCUG rGrGrGrUrGrUrUrGrGrCrCrC OOOOOOOOO rs362268GGUGUUG OOOOOO GCCC WV- CCUCCUG 859 rCrCrUrCrCrUrGrCrArGrGrCrU 1211OOOOOOOOO HTT- 959 CAGGCUG rGrGrCrUrGrUrUrGrGrCrCrC OOOOOOOOO rs362268GCUGUUG OOOOOO GCCC WV- GGGCCAA 860 G * SG * SG * SC * SC * SA * 1212SSSSSSSSSSR Stereopure PS HTT- 960 CAGCCAG SA * SC * SA * SG * SC * RC *SSSSSSSS DNA; One Rp at rs362268 CCTGCA SA * SG * SC * SC * ST * SG *position 11 SC * SA WV- GGCCAAC 861 G * SG * SC * SC * SA * SA * 1213SSSSSSSSSRS Stereopure PS HTT- 961 AGCCAGC SC * SA * SG * SC * RC * SA *SSSSSSSS DNA; One Rp at rs362268 CTGCAG SG * SC * SC * ST * SG * SC *position 10 SA * SG WV- GCCAACA 862 G * SC * SC * SA * SA * SC * 1214SSSSSSSSRSS Stereopure PS HTT- 962 GCCAGCC SA * SG * SC * RC * SA * SG *SSSSSSSS DNA; One Rp at rs362268 TGCAGG SC * SC * ST * SG * SC * SA *position 9 SG * SG WV- CCAACAG 863 C * SC * SA * SA * SC * SA * 1215SSSSSSSRSSS Stereopure PS HTT- 963 CCAGCCT SG * SC * RC * SA * SG * SC *SSSSSSSS DNA; One Rp at rs362268 GCAGGA SC * ST * SG * SC * SA * SG *position 8 SG * SA WV- CAACAGC 864 C * SA * SA * SC * SA * SG * 1216SSSSSSRSSSS Stereopure PS HTT- 964 CAGCCTG SC * RC * SA * SG * SC * SC *SSSSSSSS DNA; One Rp at rs362268 CAGGAG ST * SG * SC * SA * SG * SG *position 7 SA * SG WV- AACAGCC 865 A * SA * SC * SA * SG * SC * 1217SSSSSRSSSSS Stereopure PS HTT- 965 AGCCTGC RC * SA * SG * SC * SC * ST *SSSSSSSS DNA; One Rp at rs362268 AGGAGG SG * SC * SA * SG * SG * SA *position 6 SG * SG WV- GGCCUUU 866 rGrGrCrCrUrUrUrCrArCrUrArC 1218OOOOOOOOO siRNA (+control Htt 973 CACUACU rUrCrCrUrArCTT OOOOOOOOOfor Renilla CCUACTT OO luciferase in psiCHECK2 plasmid) antisense strandWV- GUAGGAG 867 rGrUrArGrGrArGrUrArGrUrGr 1219 OOOOOOOOO siRNA (+controlHtt SNP 974 UAGUGAA ArArArGrGrCrCTT OOOOOOOOO for Renilla rs362268AGGCCTT OO luciferase in psiCHECK2 plasmid) sense strand WV- GTAGGAG 868G * T * A * G * G * A * G * T * 1220 XXXXXXXXX ASO (+control for Htt SNP975 TAGTGAA A * G * T * G * A * A * A * G * XXXXXXXXXRenilla luciferase in rs362268 AGGCCA G * C * C * A X psiCHECK2 plasmid)WV- GCAGGGC 869 G * SC * SA * SG * SG * SG * 1221 SSSSSSSSSSSHtt seq 307 Htt 982 ACAAGGG SC * SA * SC * SA * SA * SG * SSSSSRSSexpanding 3 nt rs362307 CACAGA SG * SG * SC * SA * SC * RA * towards 3′example 3 SG * SA WV- CAGGGCA 870 C * SA * SG * SG * SG * SC * 1222SSSSSSSSSSS Htt seq 307 Htt 983 CAAGGGC SA * SC * SA * SA * SG * SG *SSSSRSSS expanding 3 nt rs362307 ACAGAC SG * SC * SA * SC * RA * SG *towards 3′ example 2 SA * SC WV- AGGGCAC 871A * SG * SG * SG * SC * SA * 1223 SSSSSSSSSSS Htt seq 307 Htt 984AAGGGCA SC * SA * SA * SG * SG * SG * SSSRSSSS expanding 3 nt rs362307CAGACT SC * SA * SC * RA * SG * SA * towards 3′ example 1 SC * ST WV-AAGGGCA 872 A * SA * SG * SG * SG * SC * 1224 SSSSSSSRSSS Htt seq 307Htt 985 CAGACTT SA * SC * RA * SG * SA * SC * SSSSSSSS expanding 3 ntrs362307 CCAAAG ST * ST * SC * SC * SA * SA * towards 5′ example 1SA * SG WV- AGGGCAC 873 A * SG * SG * SG * SC * SA * 1225 SSSSSSRSSSSHtt seq 307 Htt 986 AGACTTC SC * RA * SG * SA * SC * ST * SSSSSSSSexpanding 3 nt rs362307 CAAAGG ST * SC * SC * SA * SA * SA * towards 5′example 2 SG * SG WV- GGGCACA 874 G * SG * SG * SC * SA * SC * 1226SSSSSRSSSSS Htt seq 307 Htt 987 GACTTCC RA * SG * SA * SC * ST * ST *SSSSSSSS expanding 3 nt rs362307 AAAGGC SC * SC * SA * SA * SA * SG *towards 5′ example 3 SG * SC WV- GAGCAGC 875G * A * G * C * A * G * C * T * 1227 XXXXXXXXX All DNA; HTT- 1001TGCAACC G * C * A * A * C * C * T * G * XXXXXXXXX stereorandom PSrs362306 TGGCAA G * C * A * A X WV- AGCAGCT 876A * G * C * A * G * C * T * G * 1228 XXXXXXXXX All DNA; HTT- 1002GCAACCT C * A * A * C * C * T * G * G * XXXXXXXXX stereorandom PSrs362306 GGCAAC C * A * A * C X WV- GCAGCTG 877G * C * A * G * C * T * G * C * 1229 XXXXXXXXX All DNA; HTT- 1003CAACCTG A * A * C * C * T * G * G * C * XXXXXXXXX stereorandom PSrs362306 GCAACA A * A * C * A X WV- CAGCTGC 878C * A * G * C * T * G * C * A * 1230 XXXXXXXXX All DNA; HTT- 1004AACCTGG A * C * C * T * G * G * C * A * XXXXXXXXX stereorandom PSrs362306 CAACAA A * C * A * A X WV- AGCTGCA 879A * G * C * T * G * C * A * A * 1231 XXXXXXXXX All DNA; HTT- 1005ACCTGGC C * C * T * G * G * C * A * A * XXXXXXXXX stereorandom PSrs362306 AACAAC C * A * A * C X WV- GCTGCAA 880G * C * T * G * C * A * A * C * 1232 XXXXXXXXX All DNA; HTT- 1006CCTGGCA C * T * G * G * C * A * A * C * XXXXXXXXX stereorandom PSrs362306 ACAACC A * A * C * C X WV- GAGCAGC 881mG * mA * mG * mC * mA * G 1233 XXXXXXXXX 5-15 (2′-OMe- HTT- 1007TGCAACC * C * T * G * C * A * A * C * C XXXXXXXXX DNA); rs362306 TGGCAA* T * G * G * C * A * A X stereorandom PS WV- AGCAGCT 882mA * mG * mC * mA * mG * C 1234 XXXXXXXXX 5-15 (2′-OMe- HTT- 1008GCAACCT * T * G * C * A * A * C * C * T XXXXXXXXX DNA); rs362306 GGCAAC* G * G * C * A * A * C X stereorandom PS WV- GCAGCTG 883mG * mC * mA * mG * mC * T 1235 XXXXXXXXX 5-15 (2′-OMe- HTT- 1009CAACCTG * G * C * A * A * C * C * T * G XXXXXXXXX DNA); rs362306 GCAACA* G * C * A * A * C * A X stereorandom PS WV- CAGCUGC 884mC * mA * mG * mC * mU * G 1236 XXXXXXXXX 5-15 (2′-OMe- HTT- 1010AACCTGG * C * A * A * C * C * T * G * G XXXXXXXXX DNA); rs362306 CAACAA* C * A * A * C * A * A X stereorandom PS WV- AGCUGCA 885mA * mG * mC * mU * mG * C 1237 XXXXXXXXX 5-15 (2′-OMe- HTT- 1011ACCTGGC * A * A * C * C * T * G * G * C XXXXXXXXX DNA); rs362306 AACAAC* A * A * C * A * A * C X stereorandom PS WV- GCUGCAA 886mG * mC * mU * mG * mC * A 1238 XXXXXXXXX 5-15 (2′-OMe- HTT- 1012CCTGGCA * A * C * C * T * G * G * C * A XXXXXXXXX DNA); rs362306 ACAACC* A * C * A * A * C * C X stereorandom PS WV- GAGCAGC 887mG * mA * mG * mC * mA * G 1239 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1013TGCAACC * C * T * G * C * A * A * C * C XXXXXXXXX DNA-2′-OMe); rs362306TGGCAA * T * mG * mG * mC * mA * X stereorandom PS mA WV- AGCAGCT 888mA * mG * mC * mA * mG * C 1240 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1014GCAACCT * T * G * C * A * A * C * C * T XXXXXXXXX DNA-2′-OMe); rs362306GGCAAC * G * mG * mC * mA * mA * X stereorandom PS mC WV- GCAGCTG 889mG * mC * mA * mG * mC * T 1241 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1015CAACCTG * G * C * A * A * C * C * T * G XXXXXXXXX DNA-2′-OMe); rs362306GCAACA * G * mC * mA * mA * mC * X stereorandom PS mA WV- CAGCUGC 890mC * mA * mG * mC * mU * G 1242 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1016AACCTGG * C * A * A * C * C * T * G * G XXXXXXXXX DNA-2′-OMe); rs362306CAACAA * C * mA * mA * mC * mA * X stereorandom PS mA WV- AGCUGCA 891mA * mG * mC * mU * mG * C 1243 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1017ACCTGGC * A * A * C * C * T * G * G * C XXXXXXXXX DNA-2′-OMe); rs362306AACAAC * A * mA * mC * mA * mA * X stereorandom PS mC WV- GCUGCAA 892mG * mC * mU * mG * mC * A 1244 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1018CCTGGCA * A * C * C * T * G * G * C * A XXXXXXXXX DNA-2′-OMe); rs362306ACAACC * A * mC * mA * mA * mC * X stereorandom PS mC WV- GAGCAGC 893mG * mA * mG * mC * mA * 1245 XXXXXXXXX 7-7-6 (2′-OMe- HTT- 1019 TGCAACCmG * mC * T * G * C * A * A * XXXXXXXXX DNA-2′-OMe); rs362306 UGGCAAC * C * mU * mG * mG * mC * X stereorandom PS mA * mA WV- GAGCAGC 894mGmAmGmCmAmGmC * T * 1246 OOOOOOXXX 7-7-6 (2′-OMe- HTT- 1020 TGCAACCG * C * A * A * C * C * XXXXXOOOO DNA-2′-OMe); rs362306 UGGCAAmUmGmGmCmAmA O stereorandom PS; PO in wings WV- AGCAGCT 895mA * mG * mC * mA * mG * 1247 XXXXXXXXX 6-7-5 (2′-OMe- HTT- 1021 GCAACCTmC * T * G * C * A * A * C * C XXXXXXXXX DNA-2′-OMe); rs362306 GGCAAC* T * G * mG * mC * mA * mA X stereorandom PS * mC WV- AGCAGCT 896mAmGmCmAmGmC * T * G * 1248 OOOOOXXXX 6-7-5 (2′-OMe- HTT- 1022 GCAACCTC * A * A * C * C * T * G * XXXXXXOOO DNA-2′-OMe); rs362306 GGCAACmGmCmAmAmC O stereorandom PS; PO in the wings WV- GCAGCTG 897mG * mC * mA * mG * mC * T 1249 XXXXXXXXX 5-7-8 (2′-OMe- HTT- 1023CAACCUG * G * C * A * A * C * C * mU * XXXXXXXXX DNA-2′-OMe); rs362306GCAACA mG * mG * mC * mA * mA * X stereorandom PS mC * mA WV- GCAGCTG898 mGmCmAmGmC * T * G * C * 1250 OOOOXXXXX 5-7-8 (2′-OMe- HTT- 1024CAACCUG A * A * C * C * XXXOOOOOO DNA-2′-OMe); rs362306 GCAACAmUmGmGmCmAmAmCmA O stereorandom PS; PO in the wings WV- GAGCAGC 899mGmAmGmCmA * G * C * T * 1251 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1025TGCAACC G * C * A * A * C * C * T * XXXXXXOOO DNA-2′-OMe); rs362306TGGCAA mGmGmCmAmA O stereorandom PS; PO in the wings WV- AGCAGCT 900mAmGmCmAmG * C * T * G * 1252 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1026GCAACCT C * A * A * C * C * T * G * XXXXXXOOO DNA-2′-OMe); rs362306GGCAAC mGmCmAmAmC O stereorandom PS; PO in the wings WV- GCAGCTG 901mGmCmAmGmCT * G * C * A 1253 OOOOOXXXX 5-10-5 (2′-OMe- HTT- 1027 CAACCTG* A * C * C * T * G * G * XXXXXXOOO DNA-2′-OMe); rs362306 GCAACAmCmAmAmCmA O stereorandom PS; PO in the wings WV- CAGCUGC 902mCmAmGmCmU * G * C * A * 1254 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1028AACCTGG A * C * C * T * G * G * C * XXXXXXOOO DNA-2′-OMe); rs362306CAACAA mAmAmCmAmA O stereorandom PS; PO in the wings WV- AGCUGCA 903mAmGmCmUmG * C * A * A * 1255 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1029ACCTGGC C * C * T * G * G * C * A * XXXXXXOOO DNA-2′-OMe); rs362306AACAAC mAmCmAmAmC O stereorandom PS; PO in the wings WV- GCUGCAA 904mGmCmUmGmC * A * A * C * 1256 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1030CCTGGCA C * T * G * G * C * A * A * XXXXXXOOO DNA-2′-OMe); rs362306ACAACC mCmAmAmCmC O stereorandom PS; PO in the wings WV- GGGCCAA 905G * G * G * C * C * A * A * C * 1257 XXXXXXXXX All DNA; HTT- 1031CAGCCAG A * G * C * C * A * G * C * C * XXXXXXXXX stereorandom PSrs362268 CCTGCA T * G * C * A X WV- GGCCAAC 906G * G * C * C * A * A * C * A * 1258 XXXXXXXXX All DNA; HTT- 1032AGCCAGC G * C * C * A * G * C * C * T * XXXXXXXXX stereorandom PSrs362268 CTGCAG G * C * A * G X WV- GCCAACA 907G * C * C * A * A * C * A * G * 1259 XXXXXXXXX All DNA; HTT- 1033GCCAGCC C * C * A * G * C * C * T * G * XXXXXXXXX stereorandom PSrs362268 TGCAGG C * A * G * G X WV- CCAACAG 908C * C * A * A * C * A * G * C * 1260 XXXXXXXXX All DNA; HTT- 1034CCAGCCT C * A * G * C * C * T * G * C * XXXXXXXXX stereorandom PSrs362268 GCAGGA A * G * G * A X WV- CAACAGC 909C * A * A * C * A * G * C * C * 1261 XXXXXXXXX All DNA; HTT- 1035CAGCCTG A * G * C * C * T * G * C * A * XXXXXXXXX stereorandom PSrs362268 CAGGAG G * G * A * G X WV- AACAGCC 910A * A * C * A * G * C * C * A * 1262 XXXXXXXXX All DNA; HTT- 1036AGCCTGC G * C * C * T * G * C * A * G * XXXXXXXXX stereorandom PSrs362268 AGGAGG G * A * G * G X WV- GGGCCAA 911mG * mG * mG * mC * mC * A 1263 XXXXXXXXX 5-15 (2′-OMe- HTT- 1037CAGCCAG * A * C * A * G * C * C * A * G XXXXXXXXX DNA); rs362268 CCTGCA* C * C * T * G * C * A X stereorandom PS WV- GGCCAAC 912mG * mG * mC * mC * mA * A 1264 XXXXXXXXX 5-15 (2′-OMe- HTT- 1038AGCCAGC * C * A * G * C * C * A * G * C XXXXXXXXX DNA); rs362268 CTGCAG* C * T * G * C * A * G X stereorandom PS WV- GCCAACA 913mG * mC * mC * mA * mA * C 1265 XXXXXXXXX 5-15 (2′-OMe- HTT- 1039GCCAGCC * A * G * C * C * A * G * C * C XXXXXXXXX DNA); rs362268 TGCAGG* T * G * C * A * G * G X stereorandom PS WV- CCAACAG 914mC * mC * mA * mA * mC * A 1266 XXXXXXXXX 5-15 (2′-OMe- HTT- 1040CCAGCCT * G * C * C * A * G * C * C * T XXXXXXXXX DNA); rs362268 GCAGGA* G * C * A * G * G * A X stereorandom PS WV- CAACAGC 915mC * mA * mA * mC * mA * G 1267 XXXXXXXXX 5-15 (2′-OMe- HTT- 1041CAGCCTG * C * C * A * G * C * C * T * G XXXXXXXXX DNA); rs362268 CAGGAG* C * A * G * G * A * G X stereorandom PS WV- AACAGCC 916mA * mA * mC * mA * mG * C 1268 XXXXXXXXX 5-15 (2′-OMe- HTT- 1042AGCCTGC * C * A * G * C * C * T * G * C XXXXXXXXX DNA); rs362268 AGGAGG* A * G * G * A * G * G X stereorandom PS WV- GGGCCAA 917mG * mG * mG * mC * mC * A 1269 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1043CAGCCAG * A * C * A * G * C * C * A * G XXXXXXXXX DNA-2′-OMe); rs362268CCUGCA * C * mC * mU * mG * mC * X stereorandom PS mA WV- GGCCAAC 918mG * mG * mC * mC * mA * A 1270 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1044AGCCAGC * C * A * G * C * C * A * G * C XXXXXXXXX DNA-2′-OMe); rs362268CUGCAG * C * mU * mG * mC * mA * X stereorandom PS mG WV- GCCAACA 919mG * mC * mC * mA * mA * C 1271 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1045GCCAGCC * A * G * C * C * A * G * C * C XXXXXXXXX DNA-2′-OMe); rs362268TGCAGG * T * mG * mC * mA * mG * X stereorandom PS mG WV- CCAACAG 920mC * mC * mA * mA * mC * A 1272 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1046CCAGCCT * G * C * C * A * G * C * C * T XXXXXXXXX DNA-2′-OMe); rs362268GCAGGA * G * mC * mA * mG * mG * X stereorandom PS mA WV- CAACAGC 921mC * mA * mA * mC * mA * G 1273 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1047CAGCCTG * C * C * A * G * C * C * T * G XXXXXXXXX DNA-2′-OMe); rs362268CAGGAG * C * mA * mG * mG * mA * X stereorandom PS mG WV- AACAGCC 922mA * mA * mC * mA * mG * C 1274 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1048AGCCTGC * C * A * G * C * C * T * G * C XXXXXXXXX DNA-2′-OMe); rs362268AGGAGG * A * mG * mG * mA * mG * X stereorandom PS mG WV- GGGCCAA 923mG * mG * mG * mC * mC * 1275 XXXXXXXXX 7-7-6 (2′-OMe- HTT- 1049 CAGCCAGmA * mA * C * A * G * C * C * XXXXXXXXX DNA-2′-OMe); rs362268 CCUGCAA * G * mC * mC * mU * mG * X stereorandom PS mC * mA WV- GGGCCAA 924mGmGmGmCmCmAmA * C * 1276 OOOOOOXXX 7-7-6 (2′-OMe- HTT- 1050 CAGCCAGA * G * C * C * A * G * XXXXXOOOO DNA-2′-OMe); rs362268 CCUGCAmCmCmUmGmCmA O stereorandom PS; PO in wings WV- GGCCAAC 925mG * mG * mC * mC * mA * 1277 XXXXXXXXX 6-7-5 (2′-OMe- HTT- 1051 AGCCAGCmA * C * A * G * C * C * A * G XXXXXXXXX DNA-2′-OMe); rs362268 CUGCAG* C * C * mU * mG * mC * mA X stereorandom PS * mG WV- GGCCAAC 926mGmGmCmCmAmA * C * A * 1278 OOOOOXXXX 6-7-5 (2′-OMe- HTT- 1052 AGCCAGCG * C * C * A * G * C * C * XXXXXXOOO DNA-2′-OMe); rs362268 CUGCAGmUmGmCmAmG O stereorandom PS; PO in the wings WV- GCCAACA 927mG * mC * mC * mA * mA * C 1279 XXXXXXXXX 5-7-8 (2′-OMe- HTT- 1053GCCAGCC * A * G * C * C * A * G * mC * XXXXXXXXX DNA-2′-OMe); rs362268UGCAGG mC * mU * mG * mC * mA * X stereorandom PS mG * mG WV- GCCAACA928 mGmCmCmAmA * C * A * G * 1280 OOOOXXXXX 5-7-8 (2′-OMe- HTT- 1054GCCAGCC C * C * A * G * XXXOOOOOO DNA-2′-OMe); rs362268 UGCAGGmCmCmUmGmCmAmGmG O stereorandom PS; PO in the wings WV- GGGCCAA 929mGmGmGmCmC * A * A * C * 1281 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1055CAGCCAG A * G * C * C * A * G * C * XXXXXXOOO DNA-2′-OMe); rs362268CCUGCA mCmUmGmCmA O stereorandom PS; PO in the wings WV- GGCCAAC 930mGmGmCmCmA * A * C * A * 1282 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1056AGCCAGC G * C * C * A * G * C * C * XXXXXXOOO DNA-2′-OMe); rs362268CUGCAG mUmGmCmAmG O stereorandom PS; PO in the wings WV- GCCAACA 931mGmCmCmAmA * C * A * G * 1283 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1057GCCAGCC C * C * A * G * C * C * T * XXXXXXOOO DNA-2′-OMe); rs362268TGCAGG mGmCmAmGmG O stereorandom PS; PO in the wings WV- CCAACAG 932mCmCmAmAmC * A * G * C * 1284 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1058CCAGCCT C * A * G * C * C * T * G * XXXXXXOOO DNA-2′-OMe); rs362268GCAGGA mCmAmGmGmA O stereorandom PS; PO in the wings WV- CAACAGC 933mCmAmAmCmA * G * C * C * 1285 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1059CAGCCTG A * G * C * C * T * G * C * XXXXXXOOO DNA-2′-OMe); rs362268CAGGAG mAmGmGmAmG O stereorandom PS; PO in the wings WV- AACAGCC 934mAmAmCmAmG * C * C * A * 1286 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1060AGCCTGC G * C * C * T * G * C * A * XXXXXXOOO DNA-2′-OMe); rs362268AGGAGG mGmGmAmGmG O stereorandom PS; PO in the wings: HTT-rs362268 WV-GUAGGAG 935 mG * mU * mA * mG * mG * A * 1287 XXXXXXXXX 5-10-5 (2′-OMe-HTT- 1061 TAGTGAA * G * T * A * G * T * G * A * A XXXXXXXXX DNA-2′-OMe);rs362268 AGGCCA * A * mG * mG * mC * mC * X stereorandom PS: mA+ve Luciferase control for psiCHECK2; WV- 975 analogue WV- GUAGGAG 936mGmUmAmGmG * A * G * T * 1288 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1062TAGTGAA A * G * T * G * A * A * A * XXXXXXOOO DNA-2′-OMe); controlAGGCCA mGmGmCmCmA O stereorandom PS; PO in the wings: +ve Luciferasecontrol for psiCHECK2; WV- 975 analogue WV- GUAGGAG 937mG * mU * mA * mG * mG * A 1289 XXXXXXXXX 5-15 (2′-OMe- HTT- 1063TAGTGAA * G * T * A * G * T * G * A * A XXXXXXXXX DNA); control AGGCCA* A * G * G * C * C * A X stereorandom PS: +ve Luciferase control forpsiCHECK2; WV- 975 analogue WV- CUCUUAC 938 mC * mU * mC * mU * mU * A1290 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1064 TGTGCTG* C * T * G * T * G * C * T * G XXXXXXXXX DNA-2′-OMe); control TGGACA* T * mG * mG * mA * mC * X stereorandom PS: mA Negative Luciferasecontrol for psiCHECK2; ONT- 67 analogue WV- CUCUUAC 939mCmUmCmUmU * A * C * T * 1291 OOOOXXXXX 5-10-5 (2′-OMe- HTT- 1065TGTGCTG G * T * G * C * T * G * T * XXXXXXOOO DNA-2′-OMe); controlTGGACA mGmGmAmCmA O stereorandom PS; PO in the wings:Negative Luciferase control for psiCHECK2; ONT- 67 analogue WV- CUCUUAC940 mC * mU * mC * mU * mU * A 1292 XXXXXXXXX 5-15 (2′-OMe- HTT- 1066TGTGCTG * C * T * G * T * G * C * T * G XXXXXXXXX DNA); control TGGACA* T * G * G * A * C * A X stereorandom PS: Negative Luciferasecontrol for psiCHECK2; ONT- 67 analogue WV- GGGCACA 941G * G * G * C * A * C * A * A * 1293 XXXXXXXXX All DNA HTT- 1067 AGGGCACG * G * G * C * d2AP * C * A * XXXXXXXXX stereorandom; P13 controlAGACTT G * A * C * T * T X (2-aminopurine): rs362307; WV-904 analogueWV- GGCACAA 942 G * G * C * A * C * A * A * G * 1294 XXXXXXXXX All DNArs362307 1068 GGGCACA G * G * C * d2AP * C * A * G * XXXXXXXXXstereorandom; P12 GACTTC A * C * T * T * C X (2-aminopurine):rs362307; WV-905 analogue WV- GCACAAG 943G * C * A * C * A * A * G * G * 1295 XXXXXXXXX All DNA rs362307 1069GGCACAG G * C * d2AP * C * A * G * A * XXXXXXXXX stereorandom; P11ACTTCC C * T * T * C * C X (2-aminopurine): rs362307; WV-906 analogueWV- GGGCACA 944 G * G * G * C * A * C * A * A * 1296 XXXXXXXXX All DNArs362307 1070 AGGGCAC G * G * G * C * dDAP * C * A * XXXXXXXXXstereorandom; P13 AGACTT G * A * C * T * T X (2;6- diaminopurine):rs362307; WV-904 analogue WV- GGCACAA 945G * G * C * A * C * A * A * G * 1297 XXXXXXXXX All DNA rs362307 1071GGGCACA G * G * C * dDAP * C * A * G * XXXXXXXXX stereorandom; P12GACTTC A * C * T * T * C X (2;6- diaminopurine): rs362307; WV-905analogue WV- GCACAAG 946 G * C * A * C * A * A * G * G * 1298 XXXXXXXXXAll DNA rs362307 1072 GGCACAG G * C * dDAP * C * A * G * A * XXXXXXXXXstereorandom; P12 ACTTCC C * T * T * C * C X (2;6- diaminopurine):rs362307; WV-906 analogue WV- GAGCCUU 947 rGrArGrCrCrUrUrUrGrGrArAr 1299OOOOOOOOO wtRNA rs362307 1073 UGGAAGU GrUrCrUrGrCrGrCrCrCrUrUrGrOOOOOOOOO CUGCGCC UrGrCrCrCrUrGrCrCrU OOOOOOOOO CUUGUGC OOOOOOO CCUGCCUWV- GAGCCUU 948 rGrArGrCrCrUrUrUrGrGrArAr 1300 OOOOOOOOO muRNA rs3623071074 UGGAAGU GrUrCrUrGrUrGrCrCrCrUrUrGr OOOOOOOOO CUGUGCCUrGrCrCrCrUrGrCrCrU OOOOOOOOO CUUGUGC OOOOOOO CCUGCCU WV- CACACGG 949rCrArCrArCrGrGrGrCrArCrArG 1301 OOOOOOOOO Antisense strand: rs3623071075 GCACAGA rArCrUrUrCrCrArA OOOOOOOOO Positive control; CUUCCAA OOCurr. Bio. Vol 19 No 9; 776 WV- GGAAGUC 950 rGrGrArArGrUrCrUrGrUrGrCr1302 OOOOOOOOO Sense strand: rs362307 1076 UGUGCCC CrCrGrUrGrUrGrCrCOOOOOOOOO Positive control; GUGUGCC OO Curr. Bio. Vol 19No 9; 777: Note: incorrectly added as rGrGrArArGrUrCr UrGrUrGrCrCrCrGrUrGrUrUrCrC (SEQ ID NO: 1553) in earlier versions of databse WV-AUUAAUA 951 mA * SmU * SmU * SmA * 1303 SSSSSSSSSSS 6-10-4 (2′-OMe- HTT1077 AATTGTC SmA * SmU * SA * SA * SA * SSRSSSSS DNA-2′-OMe) rs7685686ATCACC ST * ST * SG * ST * SC * RA * Gapmer: AnalogueST * SmC * SmA * SmC * SmC of WV-451 WV- AUUAAUA 952mA * RmU * RmU * RmA * 1304 RRRRRSSSSS 6-10-4 (2′-OMe- HTT 1078 AATTGTCRmA * RmU * SA * SA * SA * SSSRSSRRR DNA-2′-OMe) rs7685686 ATCACCST * ST * SG * ST * SC * RA * Gapmer: Analogue ST * SmC * RmA * RmC *of WV-451 RmC WV- AUUAAUA 953 mA * SmU * SmU * SmA * 1305 SSSSSSSSSSS8-12 (2′-OMe- HTT 1079 AATTGTC SmA * SmU * SmA * SmA * SSRSSSSSDNA) hemimer: rs7685686 ATCACC SA * ST * ST * SG * ST * SC *Analogue of WV- RA * ST * SC * SA * SC * SC 451 WV- AUUAAUA 954mA * RmU * RmU * RmA * 1306 RRRRRRRSSS 8-12 (2′-OMe- HTT 1080 AATTGTCRmA * RmU * RmA * RmA * SSSRSSSSS DNA) hemimer: rs7685686 ATCACCSA * ST * ST * SG * ST * SC * Analogue of WV-RA * ST * SC * SA * SC * SC 451 WV- AUUAAUA 955 mAmUmUmAmAmUmAmA * 1307OOOOOOOSS 8-12 (2′-OMe- HTT 1081 AATTGTC SA * ST * ST * SG * ST * SC *SSSSRSSSSS DNA) hemimer; rs7685686 ATCACC RA * ST * SC * SA * SC * SCPO wing: Analogue of WV-451 WV- AUUAAUA 956 mAmUmUmAmAmU * SA * 1308OOOOOSSSSS 6-10-4 (2′-OMe- HTT 1082 AATTGTCSA * SA * ST * ST * SG * ST * SSSRSSOOO DNA-2′-OMe); PO rs7685686 ATCACCSC * RA * ST * SmCmAmCmC wings: Analogue of WV-451 WV- AUUAAUA 957mA * SmUmUmAmAmU * SA 1309 SOOOOSSSSS 6-10-4 (2′-OMe- HTT 1083 AATTGTC* SA * SA * ST * ST * SG * ST SSSRSSOOS DNA-2′-OMe) rs7685686 ATCACC* SC * RA * ST * SmCmAmC * Gapmer: Analogue SmC of WV-451 WV- AUUAAUA958 mA * RmUmUmAmAmU * SA 1310 ROOOOSSSSS 6-10-4 (2′-OMe- HTT 1084AATTGTC * SA * SA * ST * ST * SG * ST SSSRSSOOR DNA-2′-OMe) rs7685686ATCACC * SC * RA * ST * SmCmAmC * Gapmer: Analogue RmC of WV-451 WV-GGCACAA 959 mG * SmG * SmC * SmA * 1311 SSSSSSSSSSS 5-10-5 (2′-OMe- HTT1085 GGGCACA SmC * SA * SA * SG * SG * SG SRSSSSSS DNA-2′-OMe) rs362307GACUUC * SC * SA * SC * RA * SG * Gapmer: AnalogueSmA * SmC * SmU * SmU * of WV-905 and SmC WV-937 WV- GGCACAA 960mG * RmG * RmC * RmA * 1312 RRRRSSSSSSS 5-10-5 (2′-OMe- HTT 1086 GGGCACARmC * SA * SA * SG * SG * SRSSRRRR DNA-2′-OMe) rs362307 GACUUCSG * SC * SA * SC * RA * SG * Gapmer: Analogue SmA * RmC * RmU * RmU *of WV-905 and RmC WV-937 WV- GGCACAA 961 mGmGmCmAmC * SA * SA * 1313OOOOSSSSSS 5-10-5 (2′-OMe- HTT 1087 GGGCACASG * SG * SG * SC * SA * SC * SSRSSOOOO DNA-2′-OMe); PO rs362307 GACUUCRA * SG * SmAmCmUmUmC wings: Analogue of WV-905 and WV- 937 WV- GGCACAA962 mG * SmG * SmC * SmA * 1314 SSSSSSSSSSS 8-12 (2′-OMe- HTT 1088GGGCACA SmC * SmA * SmA * SmG * SG SRSSSSSS DNA) hemimer: rs362307GACTTC * SG * SC * SA * SC * RA * SG Analogue of WV-* SA * SC * ST * ST * SC 905 and WV-937 WV- GGCACAA 963mG * RmG * RmC * RmA * 1315 RRRRRRRSSS 8-12 (2′-OMe- HTT 1089 GGGCACARmC * RmA * RmA * RmG * SSRSSSSSS DNA) hemimer: rs362307 GACTTCSG * SG * SC * SA * SC * RA * Analogue of WV-SG * SA * SC * ST * ST * SC 905 and WV-937 WV- GGCACAA 964mGmGmCmAmCmAmAmG * 1316 OOOOOOOSS 8-12 (2′-OMe- HTT 1090 GGGCACASG * SG * SC * SA * SC * RA * SSSRSSSSSS DNA) hemimer; rs362307 GACTTCSG * SA * SC * ST * ST * SC PO wing: Analogue of WV-905 and WV-937 WV-GGCACAA 965 mG * RmGmCmAmC * SA * 1317 ROOOSSSSSS 8-12 (2′-OMe- HTT 1091GGGCACA SA * SG * SG * SG * SC * SA * SSRSSOOOR DNA) gapmer PO rs362307GACUUC SC * RA * SG * SmAmCmUmU wing: Analogue of * RmC WV-905 and WV-937: incorrectly added as gsSgcacsSdAsSdAs SdGsSdGsSdGsSd CsSdAsSdCsRdAsSdGsSacuusSc in earlier version of database WV- GGCACAA 966mG * SmGmCmAmC * SA * 1318 SOOOSSSSSS 8-12 (2′-OMe- HTT 1092 GGGCACASA * SG * SG * SG * SC * SA * SSRSSOOOS DNA) gapmer PO rs362307 GACUUCSC * RA * SG * SmAmCmUmU wing: Analogue of * SmC WV-905 and WV- 937 WV-GCAGGGC 967 G * C * A * G * G * G * C * A * 1319 XXXXXXXXXPhosphorothioate Huntington 1183 ACAAGGG C * A * A * G * G * G * C * A *XXXXXXXXX DNA; rs362307 CACAGA C * A * G * A X Stereorandom WV- GCAGGGC968 mG * mC * mA * mG * mG * G 1320 XXXXXXXXX 5-15 (2′-OMe- Huntington1184 ACAAGGG * C * A * C * A * A * G * G * G XXXXXXXXX DNA) Hemimerrs362307 CACAGA * C * A * C * A * G * A X WV- GCAGGGC 969mGmCmAmGmG * G * C * A * 1321 OOOOXXXXX 5-15 (2′-OMe- Huntington 1185ACAAGGG C * A * A * G * G * G * C * A * XXXXXXXXX DNA) Hemimer; rs362307CACAGA C * A * G * A X PO wing WV- GCAGGGC 970 mG * mC * mA * mG * mG *1322 XXXXXXXXX 7-13 (2′-OMe- Huntington 1186 ACAAGGGmG * mC * A * C * A * A * G * XXXXXXXXX DNA) Hemimer rs362307 CACAGAG * G * C * A * C * A * G * A X WV- GCAGGGC 971 mGmCmAmGmGmGmC * A *1323 OOOOOOXXX 7-13 (2′-OMe- Huntington 1187 ACAAGGGC * A * A * G * G * G * C * A * XXXXXXXXX DNA) Hemimer; rs362307 CACAGAC * A * G * A X PO wing WV- CAGGGCA 972 C * A * G * G * G * C * A * C *1324 XXXXXXXXX Phosphorothioate Huntington 1188 CAAGGGCA * A * G * G * G * C * A * C * XXXXXXXXX DNA; rs362307 ACAGACA * G * A * C X Stereorandom WV- CAGGGCA 973 mC * mA * mG * mG * mG * C1325 XXXXXXXXX 5-15 (2′-OMe- Huntington 1189 CAAGGGC* A * C * A * A * G * G * G * C XXXXXXXXX DNA) Hemimer rs362307 ACAGAC* A * C * A * G * A * C X WV- CAGGGCA 974 mCmAmGmGmG * C * A * C * 1326OOOOXXXXX 5-15 (2′-OMe- Huntington 1190 CAAGGGCA * A * G * G * G * C * A * C * XXXXXXXXX DNA) Hemimer; rs362307 ACAGACA * G * A * C X PO wing WV- CAGGGCA 975 mC * mA * mG * mG * mG * 1327XXXXXXXXX 7-13 (2′-OMe- Huntington 1191 CAAGGGCmC * mA * C * A * A * G * G * XXXXXXXXX DNA) Hemimer rs362307 ACAGACG * C * A * mC * mA * mG * X mA * mC WV- CAGGGCA 976mCmAmGmGmGmCmA * C * 1328 OOOOOOXXX 7-13 (2′-OMe- Huntington 1192CAAGGGC A * A * G * G * G * C * A * XXXXXXOOO DNA) Hemimer; rs362307ACAGAC mCmAmGmAmC O PO wing WV- AGGGCAC 977A * G * G * G * C * A * C * A * 1329 XXXXXXXXX PhosphorothioateHuntington 1193 AAGGGCA A * G * G * G * C * A * C * A * XXXXXXXXX DNA;rs362307 CAGACT G * A * C * T X Stereorandom WV- AGGGCAC 978mA * mG * mG * mG * mC * A 1330 XXXXXXXXX 5-15 (2′-OMe- Huntington 1194AAGGGCA * C * A * A * G * G * G * C * A XXXXXXXXX DNA) Hemimer rs362307CAGACT * C * A * G * A * C * T X WV- AGGGCAC 979mAmGmGmGmC * A * C * A * 1331 OOOOXXXXX 5-15 (2′-OMe- Huntington 1195AAGGGCA A * G * G * G * C * A * C * A * XXXXXXXXX DNA) Hemimer; rs362307CAGACT G * A * C * T X PO wing WV- AGGGCAC 980 mA * mG * mG * mG * mC *1332 XXXXXXXXX 7-12-1 (2′-OMe- Huntington 1196 AAGGGCAmA * mC * A * A * G * G * G * XXXXXXXXX DNA-2′-DNA) rs362307 CAGACUC * A * C * A * G * A * C * mU X Gapmer WV- AGGGCAC 981mAmGmGmGmCmAmC * A * 1333 OOOOOOXXX 7-12-1 (2′-OMe- Huntington 1197AAGGGCA A * G * G * G * C * A * C * A * XXXXXXXXX DNA-2′-DNA) rs362307CAGACU G * A * C * mU X Gapmer; PO wings WV- AAGGGCA 982A * A * G * G * G * C * A * C * 1334 XXXXXXXXX PhosphorothioateHuntington 1198 CAGACTT A * G * A * C * T * T * C * C * XXXXXXXXX DNA;rs362307 CCAAAG A * A * A * G X Stereorandom WV- AAGGGCA 983mA * mA * mG * mG * mG * C 1335 XXXXXXXXX 5-15 (2′-OMe- Huntington 1199CAGACTT * A * C * A * G * A * C * T * T XXXXXXXXX DNA) Hemimer rs362307CCAAAG * C * C * A * A * A * G X WV- AAGGGCA 984mAmAmGmGmG * C * A * C * 1336 OOOOXXXXX 5-15 (2′-OMe- Huntington 1200CAGACTT A * G * A * C * T * T * C * C * XXXXXXXXX DNA) Hemimer; rs362307CCAAAG A * A * A * G X PO wing WV- AAGGGCA 985mA * mA * mG * mG * mG * C 1337 XXXXXXXXX 5-10-5 (2′-OMe- Huntington1201 CAGACTT * A * C * A * G * A * C * T * T XXXXXXXXX DNA-2′-DNA)rs362307 CCAAAG * C * mC * mA * mA * mA * X Gapmer mG WV- AAGGGCA 986mAmAmGmGmG * C * A * C * 1338 OOOOXXXXX 5-10-5 (2′-OMe- Huntington 1202CAGACTT A * G * A * C * T * T * C * XXXXXXOOO DNA-2′-DNA) rs362307CCAAAG mCmAmAmAmG O Gapmer; PO wings WV- AAGGGCA 987mA * mA * mG * mG * G * C * 1339 XXXXXXXXX 4-10-6 (2′-OMe- Huntington1203 CAGACTT A * C * A * G * A * C * T * T * XXXXXXXXX DNA-2′-DNA)rs362307 CCAAAG mC * mC * mA * mA * mA * X Gapmer mG WV- AAGGGCA 988mAmAmGmGG * C * A * C * 1340 OOOOXXXXX 4-10-6 (2′-OMe- Huntington 1204CAGACTT A * G * A * C * T * T * XXXXXOOOO DNA-2′-DNA) rs362307 CCAAAGmCmCmAmAmAmG O Gapmer; PO wings WV- AGGGCAC 989A * G * G * G * C * A * C * A * 1341 XXXXXXXXX PhosphorothioateHuntington 1205 AGACTTC G * A * C * T * T * C * C * A * XXXXXXXXX DNA;rs362307 CAAAGG A * A * G * G X Stereorandom WV- AGGGCAC 990mA * mG * mG * mG * mC * A 1342 XXXXXXXXX 5-15 (2′-OMe- Huntington 1206AGACTTC * C * A * G * A * C * T * T * C XXXXXXXXX DNA) Hemimer rs362307CAAAGG * C * A * A * A * G * G X WV- AGGGCAC 991mAmGmGmGmC * A * C * A * 1343 OOOOXXXXX 5-15 (2′-OMe- Huntington 1207AGACTTC G * A * C * T * T * C * C * A * XXXXXXXXX DNA) Hemimer; rs362307CAAAGG A * A * G * G X PO wing WV- AGGGCAC 992mA * mG * mG * mG * mC * A 1344 XXXXXXXXX 5-10-5 (2′-OMe- Huntington1208 AGACTTC * C * A * G * A * C * T * T * C XXXXXXXXX DNA-2′-DNA)rs362307 CAAAGG * C * mA * mA * mA * mG * X Gapmer mG WV- AGGGCAC 993mAmGmGmGmC * A * C * A * 1345 OOOOXXXXX 5-10-5 (2′-OMe- Huntington 1209AGACTTC G * A * C * T * T * C * C * XXXXXXOOO DNA-2′-DNA) rs362307CAAAGG mAmAmAmGmG O Gapmer; PO wings WV- AGGGCAC 994mA * mG * mG * mG * C * A * 1346 XXXXXXXXX 4-10-6 (2′-OMe- Huntington1210 AGACTTC C * A * G * A * C * T * T * C * XXXXXXXXX DNA-2′-DNA)rs362307 CAAAGG mC * mA * mA * mA * mG * X Gapmer mG WV- AGGGCAC 995mAmGmGmG * C * A * C * A 1347 OOOXXXXXX 4-10-6 (2′-OMe- Huntington 1211AGACTTC * G * A * C * T * T * C * XXXXXOOOO DNA-2′-DNA) rs362307 CAAAGGmCmAmAmAmGmG O Gapmer; PO wings WV- GGGCACA 996G * G * G * C * A * C * A * G * 1348 XXXXXXXXX PhosphorothioateHuntington 1212 GACTTCC A * C * T * T * C * C * A * A * XXXXXXXXX DNA;rs362307 AAAGGC A * G * G * C X Stereorandom WV- GGGCACA 997mG * mG * mG * mC * mA * C 1349 XXXXXXXXX 4-16 (2′-OMe- Huntington 1213GACTTCC * A * G * A * C * T * T * C * C XXXXXXXXX DNA) Hemimer rs362307AAAGGC * A * A * A * G * G * C X WV- GGGCACA 998mGmGmGmCmA * C * A * G * 1350 OOOOXXXXX 4-16 (2′-OMe- Huntington 1214GACTTCC A * C * T * T * C * C * A * A * XXXXXXXXX DNA) Hemimer; rs362307AAAGGC A * G * G * C X PO wing WV- GGGCACA 999mG * mG * mG * mC * mA * C 1351 XXXXXXXXX 4-10-6 (2′-OMe- Huntington1215 GACTTCC * A * G * A * C * T * T * C * C XXXXXXXXX DNA-2′-DNA)rs362307 AAAGGC * A * mA * mA * mG * mG * X Gapmer mC WV- GGGCACA 1000mGmGmGmCmA * C * A * G * 1352 OOOOXXXXX 4-10-6 (2′-OMe- Huntington 1216GACTTCC A * C * T * T * C * C * A * XXXXXXOOO DNA-2′-DNA) rs362307AAAGGC mAmAmGmGmC O Gapmer; PO wings WV- GGCACAA 1001mG * mG * mC * mA * mC * A 1353 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1234GGGCACA * A * G * G * G * C * A * C * A XXXXXXXXX DNA-2′-OMe) rs362307GACUTC * G * mA * mC * mU * BrdU * X Gapmer; One Br- mC dU WV- GGCACAA1002 mG * mG * mC * mA * mC * A 1354 XXXXXXXXX 5-10-5 (2′-OMe- HTT- 1235GGGCACA * A * G * G * G * C * A * C * A XXXXXXXXX DNA-2′-OMe) rs362307GACTTC * G * mA * mC * BrdU * BrdU X Gapmer; two Br-dU * mC WV- GGCACAA1003 mG * mGmCmAmC * A * A * 1355 XOOOXXXXX stereo random HTT 1497GGGCACA G * G * G * C * A * C * A * G * XXXXXXOOO version of WV-rs362307 GACUUC mAmCmUmU * mC X 1092 WV- AUUAAUA 1004A * SmUmUmAmAmU * SA * 1356 SOOOOSSSSS 1-5-10-3-1 HTT 1508 AATTGTCSA * SA * ST * ST * SG * ST * SSSRSSOOS (DNA/2′-OMe) rs7685686 ATCACCSC * RA * ST * SmCmAmC * Gapmer: : SC Analogue of WV- 1083 WV- AUUAAUA1005 A * mUmUmAmAmU * A * A * 1357 XOOOOXXXX 1-5-10-3-1 HTT 1509 AATTGTCA * T * T * G * T * C * A * T * XXXXXXXOO (DNA/2′-OMe) rs7685686 ATCACCmCmAmC * C X Gapmer; 1st and last PS: : Analogue of WV-1083 WV- GGCACAA1006 G * SmGmCmAmC * SA * SA * 1358 SOOOSSSSSS 1-4-10-4-1 HTT 1510GGGCACA SG * SG * SG * SC * SA * SC * SSRSSOOOS (DNA/2′-OMe) rs362307GACUUC RA * SG * SmAmCmUmU * SC gapmer: : Analogue of WV-1092 WV-GGCACAA 1007 G * mGmCmAmC * A * A * G 1359 XOOOXXXXX 1-4-10-4-1 HTT 1511GGGCACA * G * G * C * A * C * A * G * XXXXXXOOO (DNA/2′-OMe) rs362307GACUUC mAmCmUmU * C X gapmer; 1st and last PS: : Analogue of WV-1092 WV-GGCACAA 1008 Geo * Geo * m5Ceo * Aeo * 1360 XXXXXXXXX 5-10-5; 2′-OMOEHTT 1654 GGGCACA m5Ceo * A * A * G * G * G * C XXXXXXXXX gapmer; All PSrs362307 GACTTC * A * C * A * G * Aeo * m5Ceo X * Teo * Teo * m5Ceo WV-GGCACAA 1009 Geo * Geom5CeoAeom5Ceo * A 1361 XOOOXXXXX 5-10-5; 2′-OMOEHTT 1655 GGGCACA * A * G * G * G * C * A * C * A XXXXXXOOOgapmer; 1st and last rs362307 GACTTC * G * Aeom5CeoTeoTeo * XPS in the wing; rest m5Ceo of the wing is PO WV- CTCAGTA 1010m5Ceo * Teo * m5Ceo * Aeo * 1362 XXXXXXXXX 5-10-5; 2′-OMOE Huntington1656 ACATTGA Geo * T * A * A * C * A * T * T XXXXXXXXX gapmer; All PSCACCAC * G * A * C * Aeo * m5Ceo * X m5Ceo * Aeo * m5Ceo WV- CUCAGTA1011 mC * mU * mC * mA * mG * T 1363 XXXXXXXXX 5-10-5; 2′-OMe Huntington1657 ACATTGA * A * A * C * A * T * T * G * A XXXXXXXXX gapmer; All PSCACCAC * C * mA * mC * mC * mA * X mC WV- GGCACAA 1012mG * mGmCmAmC * A * A * 1364 XOOOXXXXX 5/10/5 2′Ome HTT 1788 GGGCACAG * G * G * C * A * C * A * G * XXXXXXOOX Gapmer BrdU PO GACUTCmAmCmU * BrdU * mC X wings WV- CTCAGTA 1013 mC * BrdU * mC * mA * mG *1365 XXXXXXXXX 5/10/5 2′Ome HTT 1789 ACATTGAT * A * A * C * A * T * T * G * XXXXXXXXX Gapmer BrdU CACCACA * C * mA * mC * mC * mA * X mC WV- CTCAGTA 1014mC * BrdU * mCmAmG * T * A 1366 XXOOXXXXX 5/10/5 2′Ome HTT 1790 ACATTGA* A * C * A * T * T * G * A * C XXXXXXOOO Gapmer BrdU PO CACCAC* mAmCmCmA * mC X wings WV- GAAGUCU 1015 rGrArArGrUrCrUrGrUrGrCrCrC 1367OOOOOOOOO RNA HTT 1799 GUGCCCU rUrUrGrUrGrCrC OOOOOOOOO complementary toUGUGCC O WV1092 WV- GGCACAA 1016 mG * SmGmCmAmC * SA * 1368 SOOOSSSSSSBrdU version of HTT 2022 GGGCACA SA * SG * SG * SG * SC * SA * SSRSSOOSSWV-1092 rs362307 GACUTC SC * RA * SG * SmAmCmU * SBrdU * SmC WV- TGTCATC1017 T * G * T * C * A * T * C * A * 1369 XXXXXXXXX 15-5 hemimer fullrs7685686 2023 ACCAGAA C * C * A * G * A * A * A * mA XXXXXXXXX PS (A/G)AAAGUC * mA * mG * mU * mC X WV- UTGTCAT 1018mU * T * G * T * C * A * T * C 1370 XXXXXXXXX 1-14-5 gapmer fullrs7685686 2024 CACCAGA * A * C * C * A * G * A * A * XXXXXXXXX PS (A/G)AAAAGU mA * mA * mA * mG * mU X WV- TTGTCAT 1019T * T * G * T * C * A * T * C * 1371 XXXXXXXXX 15-5 hemimer fullrs7685686 2025 CACCAGA A * C * C * A * G * A * A * mA XXXXXXXXX PS (A/G)AAAAGU * mA * mA * mG * mU X WV- AUTGTCA 1020mA * mU * T * G * T * C * A * 1372 XXXXXXXXX 2-13-5 gapmer fullrs7685686 2026 TCACCAG T * C * A * C * C * A * G * A * XXXXXXXXX PS(A/G) AAAAAG mA * mA * mA * mA * mG X WV- ATTGTCA 1021mA * T * T * G * T * C * A * T 1373 XXXXXXXXX 1-14-5 gapmer fullrs7685686 2027 TCACCAG * C * A * C * C * A * G * A * XXXXXXXXX PS (A/G)AAAAAG mA * mA * mA * mA * mG X WV- AAUTGTC 1022mA * mA * mU * T * G * T * C 1374 XXXXXXXXX 3-12-5 gapmer full rs76856862028 ATCACCA * A * T * C * A * C * C * A * G XXXXXXXXX PS (A/G) GAAAAA* mA * mA * mA * mA * mA X WV- AATTGTC 1023mA * mA * T * T * G * T * C * 1375 XXXXXXXXX 2-13-5 gapmer fullrs7685686 2029 ATCACCA A * T * C * A * C * C * A * G * XXXXXXXXX PS(A/G) GAAAAA mA * mA * mA * mA * mA X WV- AAATTGT 1024mA * mA * mA * T * T * G * T 1376 XXXXXXXXX 3-12-5 gapmer full rs76856862030 CATCACC * C * A * T * C * A * C * C * A XXXXXXXXX PS (A/G) AGAAAA* mG * mA * mA * mA * mA X WV- AAAUTGT 1025 mA * mA * mA * mU * T * G *1377 XXXXXXXXX 4-11-5 gapmer full rs7685686 2031 CATCACCT * C * A * T * C * A * C * C * XXXXXXXXX PS (A/G) AGAAAAA * mG * mA * mA * mA * mA X WV- UAAAUTG 1026 mU * mA * mA * mA * mU * T1378 XXXXXXXXX 5-11-4 gapmer full rs7685686 2032 TCATCAC* G * T * C * A * T * C * A * C XXXXXXXXX PS (A/G) CAGAAA* C * A * mG * mA * mA * mA X WV- UAAAUTG 1027mU * mA * mA * mA * mU * T 1379 XXXXXXXXX 5-10-5 gapmer full rs76856862033 TCATCAC * G * T * C * A * T * C * A * C XXXXXXXXX PS (A/G) CAGAAA* C * mA * mG * mA * mA * X mA WV- AUAAATT 1028mA * mU * mA * mA * mA * T 1380 XXXXXXXXX 5-11-4 gapmer full rs76856862034 GTCATCA * T * G * T * C * A * T * C * A XXXXXXXXX PS (A/G) CCAGAA* C * C * mA * mG * mA * mA X WV- AUAAATT 1029mA * mU * mA * mA * mA * T 1381 XXXXXXXXX 5-10-5 gapmer full rs76856862035 GTCATCA * T * G * T * C * A * T * C * A XXXXXXXXX PS (A/G) CCAGAA* C * mC * mA * mG * mA * X mA WV- AAUAAAT 1030mA * mA * mU * mA * mA * A 1382 XXXXXXXXX 5-12-3 gapmer full rs76856862036 TGTCATC * T * T * G * T * C * A * T * C XXXXXXXXX PS (A/G) ACCAGA* A * C * C * mA * mG * mA X WV- AAUAAAT 1031 mA * mA * mU * mA * mA * A1383 XXXXXXXXX 5-11-4 gapmer full rs7685686 2037 TGTCATC* T * T * G * T * C * A * T * C XXXXXXXXX PS (A/G) ACCAGA* A * C * mC * mA * mG * mA X WV- AAUAAAT 1032mA * mA * mU * mA * mA * A 1384 XXXXXXXXX 5-10-5 gapmer full rs76856862038 TGTCATC * T * T * G * T * C * A * T * C XXXXXXXXX PS (A/G) ACCAGA* A * mC * mC * mA * mG * X mA WV- UAAUAAA 1033mU * mA * mA * mU * mA * A 1385 XXXXXXXXX 5-13-2 gapmer full rs76856862039 TTGTCAT * A * T * T * G * T * C * A * T XXXXXXXXX PS (A/G) CACCAG* C * A * C * C * mA * mG X WV- UAAUAAA 1034 mU * mA * mA * mU * mA * A1386 XXXXXXXXX 5-12-3 gapmer full rs7685686 2040 TTGTCAT* A * T * T * G * T * C * A * T XXXXXXXXX PS (A/G) CACCAG* C * A * C * mC * mA * mG X WV- UAAUAAA 1035 mU * mA * mA * mU * mA * A1387 XXXXXXXXX 5-11-4 gapmer full rs7685686 2041 TTGTCAT* A * T * T * G * T * C * A * T XXXXXXXXX PS (A/G) CACCAG* C * A * mC * mC * mA * mG X WV- UAAUAAA 1036mU * mA * mA * mU * mA * A 1388 XXXXXXXXX 5-10-5 gapmer full rs76856862042 TTGTCAT * A * T * T * G * T * C * A * T XXXXXXXXX PS (A/G) CACCAG* C * mA * mC * mC * mA * X mG WV- UUAAUAA 1037mU * mU * mA * mA * mU * A 1389 XXXXXXXXX 5-14-1 gapmer full rs76856862043 ATTGTCA * A * A * T * T * G * T * C * A XXXXXXXXX PS (A/G) TCACCA* T * C * A * C * C * mA X WV- UUAAUAA 1038 mU * mU * mA * mA * mU * A1390 XXXXXXXXX 5-13-2 gapmer full rs7685686 2044 ATTGTCA* A * A * T * T * G * T * C * A XXXXXXXXX PS (A/G) TCACCA* T * C * A * C * mC * mA X WV- UUAAUAA 1039 mU * mU * mA * mA * mU * A1391 XXXXXXXXX 5-12-3 gapmer full rs7685686 2045 ATTGTCA* A * A * T * T * G * T * C * A XXXXXXXXX PS (A/G) TCACCA* T * C * A * mC * mC * mA X WV- UUAAUAA 1040 mU * mU * mA * mA * mU * A1392 XXXXXXXXX 5-11-4 gapmer full rs7685686 2046 ATTGTCA* A * A * T * T * G * T * C * A XXXXXXXXX PS (A/G) TCACCA* T * C * mA * mC * mC * mA X WV- AUUAATA 1041mA * mU * mU * mA * mA * T 1393 XXXXXXXXX 5-15 hemimer full rs76856862047 AATTGTC * A * A * A * T * T * G * T * C XXXXXXXXX PS (A/G) ATCACC* A * T * C * A * C * C X WV- AUUAATA 1042 mA * mU * mU * mA * mA * T1394 XXXXXXXXX 5-14-1 gapmer full rs7685686 2048 AATTGTC* A * A * A * T * T * G * T * C XXXXXXXXX PS (A/G) ATCACC* A * T * C * A * C * mC X WV- AUUAATA 1043 mA * mU * mU * mA * mA * T1395 XXXXXXXXX 5-13-2 gapmer full rs7685686 2049 AATTGTC* A * A * A * T * T * G * T * C XXXXXXXXX PS (A/G) ATCACC* A * T * C * A * mC * mC X WV- AUUAATA 1044 mA * mU * mU * mA * mA * T1396 XXXXXXXXX 5-12-3 gapmer full rs7685686 2050 AATTGTC* A * A * A * T * T * G * T * C XXXXXXXXX PS (A/G) ATCACC* A * T * C * mA * mC * mC X WV- UAUUAAT 1045 mU * mA * mU * mU * mA * A1397 XXXXXXXXX 5-15 hemimer full rs7685686 2051 AAATTGT* T * A * A * A * T * T * G * T XXXXXXXXX PS (A/G) CATCAC* C * A * T * C * A * C X WV- UAUUAAT 1046 mU * mA * mU * mU * mA * A1398 XXXXXXXXX 5-14-1 gapmer full rs7685686 2052 AAATTGT* T * A * A * A * T * T * G * T XXXXXXXXX PS (A/G) CATCAC* C * A * T * C * A * mC X WV- UAUUAAT 1047 mU * mA * mU * mU * mA * A1399 XXXXXXXXX 5-13-2 gapmer full rs7685686 2053 AAATTGT* T * A * A * A * T * T * G * T XXXXXXXXX PS (A/G) CATCAC* C * A * T * C * mA * mC X WV- CUAUUAA 1048 mC * mU * mA * mU * mU * A1400 XXXXXXXXX 5-15 hemimer full rs7685686 2054 TAAATTG* A * T * A * A * A * T * T * G XXXXXXXXX PS (A/G) TCATCA* T * C * A * T * C * A X WV- CUAUUAA 1049 mC * mU * mA * mU * mU * A1401 XXXXXXXXX 5-14-1 gapmer full rs7685686 2055 TAAATTG* A * T * A * A * A * T * T * G XXXXXXXXX PS (A/G) TCATCA* T * C * A * T * C * mA X WV- ACUAUTA 1050 mA * mC * mU * mA * mU * T1402 XXXXXXXXX 5-15 hemimer full rs7685686 2056 ATAAATT* A * A * T * A * A * A * T * T XXXXXXXXX PS (A/G) GTCATC* G * T * C * A * T * C X WV- TGTCATC 1051T * G * T * C * A * T * C * A * 1403 XXXXXXXXX 15-5 hemimer 1 PSrs7685686 2057 ACCAGAA C * C * A * G * A * A * A * XXXXXXOOOon each end and (A/G) AAAGUC mAmAmGmU * mC X between dN-mN and dN-dN WV-UTGTCAT 1052 mU * T * G * T * C * A * T * C 1404 XXXXXXXXX1-14-5 gapmer 1 rs7685686 2058 CACCAGA * A * C * C * A * G * A * A *XXXXXXOOO PS on each end and (A/G) AAAAGU mAmAmAmG * mU X between dN-mNand dN-dN WV- TTGTCAT 1053 T * T * G * T * C * A * T * C * 1405XXXXXXXXX 15-5 hemimer 1 PS rs7685686 2059 CACCAGAA * C * C * A * G * A * A * XXXXXXOOO on each end and (A/G) AAAAGUmAmAmAmG * mU X between dN-mN and dN-dN WV- AUTGTCA 1054mA * mU * T * G * T * C * A * 1406 XXXXXXXXX 2-13-5 gapmer 1 rs76856862060 TCACCAG T * C * A * C * C * A * G * A * XXXXXXOOOPS on each end and (A/G) AAAAAG mAmAmAmA * mG X between dN-mN and dN-dNWV- ATTGTCA 1055 mA * T * T * G * T * C * A * T 1407 XXXXXXXXX1-14-5 gapmer 1 rs7685686 2061 TCACCAG * C * A * C * C * A * G * A *XXXXXXOOO PS on each end and (A/G) AAAAAG mAmAmAmA * mG X between dN-mNand dN-dN WV- AAUTGTC 1056 mA * mAmU * T * G * T * C * 1408 XOXXXXXXX3-12-5 gapmer 1 rs7685686 2062 ATCACCA A * T * C * A * C * C * A * G *XXXXXXOOO PS on each end and (A/G) GAAAAA mAmAmAmA * mA X between dN-mNand dN-dN WV- AATTGTC 1057 mA * mA * T * T * G * T * C * 1409 XXXXXXXXX2-13-5 gapmer 1 rs7685686 2063 ATCACCA A * T * C * A * C * C * A * G *XXXXXXOOO PS on each end and (A/G) GAAAAA mAmAmAmA * mA X between dN-mNand dN-dN WV- AAATTGT 1058 mA * mAmA * T * T * G * T * 1410 XOXXXXXXX3-12-5 gapmer 1 rs7685686 2064 CATCACC C * A * T * C * A * C * C * A *XXXXXXOOO PS on each end and (A/G) AGAAAA mGmAmAmA * mA X between dN-mNand dN-dN WV- AAAUTGT 1059 mA * mAmAmU * T * G * T * 1411 XOOXXXXXX4-11-5 gapmer 1 rs7685686 2065 CATCACC C * A * T * C * A * C * C * A *XXXXXXOOO PS on each end and (A/G) AGAAAA mGmAmAmA * mA X between dN-mNand dN-dN WV- UAAAUTG 1060 mU * mAmAmAmU * T * G * T 1412 XOOOXXXXX5-11-4 gapmer 1 rs7685686 2066 TCATCAC * C * A * T * C * A * C * C * AXXXXXXXOO PS on each end and (A/G) CAGAAA * mGmAmA * mA X between dN-mNand dN-dN WV- UAAAUTG 1061 mU * mAmAmAmU * T * G * T 1413 XOOOXXXXX5-10-5 gapmer 1 rs7685686 2067 TCATCAC * C * A * T * C * A * C * C *XXXXXXOOO PS on each end and (A/G) CAGAAA mAmGmAmA * mA X between dN-mNand dN-dN WV- AUAAATT 1062 mA * mUmAmAmA * T * T * G 1414 XOOOXXXXX5-11-4 gapmer 1 rs7685686 2068 GTCATCA * T * C * A * T * C * A * C * CXXXXXXXOO PS on each end and (A/G) CCAGAA * mAmGmA * mA X between dN-mNand dN-dN WV- AUAAATT 1063 mA * mUmAmAmA * T * T * G 1415 XOOOXXXXX5-10-5 gapmer 1 rs7685686 2069 GTCATCA * T * C * A * T * C * A * C *XXXXXXOOO PS on each end and (A/G) CCAGAA mCmAmGmA * mA X between dN-mNand dN-dN WV- AAUAAAT 1064 mA * mAmUmAmA * A * T * T 1416 XOOOXXXXX5-12-3 gapmer 1 rs7685686 2070 TGTCATC * G * T * C * A * T * C * A * CXXXXXXXXO PS on each end and (A/G) ACCAGA * C * mAmG * mA Xbetween dN-mN and dN-dN WV- AAUAAAT 1065 mA * mAmUmAmA * A * T * T 1417XOOOXXXXX 5-11-4 gapmer 1 rs7685686 2071 TGTCATC* G * T * C * A * T * C * A * C XXXXXXXOO PS on each end and (A/G)ACCAGA * mCmAmG * mA X between dN-mN and dN-dN WV- AAUAAAT 1066mA * mAmUmAmA * A * T * T 1418 XOOOXXXXX 5-10-5 gapmer 1 rs7685686 2072TGTCATC * G * T * C * A * T * C * A * XXXXXXOOO PS on each end and (A/G)ACCAGA mCmCmAmG * mA X between dN-mN and dN-dN WV- UAAUAAA 1067mU * mAmAmUmA * A * A * 1419 XOOOXXXXX 5-13-2 gapmer 1 rs7685686 2073TTGTCAT T * T * G * T * C * A * T * C * XXXXXXXXX PS on each end and(A/G) CACCAG A * C * C * mA * mG X between dN-mN and dN-dN WV- UAAUAAA1068 mU * mAmAmUmA * A * A * 1420 XOOOXXXXX 5-12-3 gapmer 1 rs76856862074 TTGTCAT T * T * G * T * C * A * T * C * XXXXXXXXOPS on each end and (A/G) CACCAG A * C * mCmA * mG X between dN-mNand dN-dN WV- UAAUAAA 1069 mU * mAmAmUmA * A * A * 1421 XOOOXXXXX5-11-4 gapmer 1 rs7685686 2075 TTGTCAT T * T * G * T * C * A * T * C *XXXXXXXOO PS on each end and (A/G) CACCAG A * mCmCmA * mG Xbetween dN-mN and dN-dN WV- UAAUAAA 1070 mU * mAmAmUmA * A * A * 1422XOOOXXXXX 5-10-5 gapmer 1 rs7685686 2076 TTGTCATT * T * G * T * C * A * T * C * XXXXXXOOO PS on each end and (A/G)CACCAG mAmCmCmA * mG X between dN-mN and dN-dN WV- UUAAUAA 1071mU * mUmAmAmU * A * A * 1423 XOOOXXXXX 5-14-1 gapmer 1 rs7685686 2077ATTGTCA A * T * T * G * T * C * A * T * XXXXXXXXX PS on each end and(A/G) TCACCA C * A * C * C * mA X between dN-mN and dN-dN WV- UUAAUAA1072 mU * mUmAmAmU * A * A * 1424 XOOOXXXXX 5-13-2 gapmer 1 rs76856862078 ATTGTCA A * T * T * G * T * C * A * T * XXXXXXXXXPS on each end and (A/G) TCACCA C * A * C * mC * mA X between dN-mNand dN-dN WV- UUAAUAA 1073 mU * mUmAmAmU * A * A * 1425 XOOOXXXXX5-12-3 gapmer 1 rs7685686 2079 ATTGTCA A * T * T * G * T * C * A * T *XXXXXXXXO PS on each end and (A/G) TCACCA C * A * mCmC * mA Xbetween dN-mN and dN-dN WV- UUAAUAA 1074 mU * mUmAmAmU * A * A * 1426XOOOXXXXX 5-11-4 gapmer 1 rs7685686 2080 ATTGTCAA * T * T * G * T * C * A * T * XXXXXXXOO PS on each end and (A/G)TCACCA C * mAmCmC * mA X between dN-mN and dN-dN WV- AUUAATA 1075mA * mUmUmAmA * T * A * 1427 XOOOXXXXX 5-15 hemimer 1 PS rs7685686 2081AATTGTC A * A * T * T * G * T * C * A * XXXXXXXXX on each end and (A/G)ATCACC T * C * A * C * C X between dN-mN and dN-dN WV- AUUAATA 1076mA * mUmUmAmA * T * A * 1428 XOOOXXXXX 5-14-1 gapmer 1 rs7685686 2082AATTGTC A * A * T * T * G * T * C * A * XXXXXXXXX PS on each end and(A/G) ATCACC T * C * A * C * mC X between dN-mN and dN-dN WV- AUUAATA1077 mA * mUmUmAmA * T * A * 1429 XOOOXXXXX 5-13-2 gapmer 1 rs76856862083 AATTGTC A * A * T * T * G * T * C * A * XXXXXXXXXPS on each end and (A/G) ATCACC T * C * A * mC * mC X between dN-mNand dN-dN WV- AUUAATA 1078 mA * mUmUmAmA * T * A * 1430 XOOOXXXXX5-12-3 gapmer 1 rs7685686 2084 AATTGTC A * A * T * T * G * T * C * A *XXXXXXXXO PS on each end and (A/G) ATCACC T * C * mAmC * mC Xbetween dN-mN and dN-dN WV- UAUUAAT 1079 mU * mAmUmUmA * A * T * 1431XOOOXXXXX 5-15 hemimer 1 PS rs7685686 2085 AAATTGTA * A * A * T * T * G * T * C * XXXXXXXXX on each end and (A/G) CATCACA * T * C * A * C X between dN-mN and dN-dN WV- UAUUAAT 1080mU * mAmUmUmA * A * T * 1432 XOOOXXXXX 5-14-1 gapmer 1 rs7685686 2086AAATTGT A * A * A * T * T * G * T * C * XXXXXXXXX PS on each end and(A/G) CATCAC A * T * C * A * mC X between dN-mN and dN-dN WV- UAUUAAT1081 mU * mAmUmUmA * A * T * 1433 XOOOXXXXX 5-13-2 gapmer 1 rs76856862087 AAATTGT A * A * A * T * T * G * T * C * XXXXXXXXXPS on each end and (A/G) CATCAC A * T * C * mA * mC X between dN-mNand dN-dN WV- CUAUUAA 1082 mC * mUmAmUmU * A * A * T 1434 XOOOXXXXX5-15 hemimer 1 PS rs7685686 2088 TAAATTG * A * A * A * T * T * G * T * CXXXXXXXXX on each end and (A/G) TCATCA * A * T * C * A X between dN-mNand dN-dN WV- CUAUUAA 1083 mC * mUmAmUmU * A * A * T 1435 XOOOXXXXX5-14-1 gapmer 1 rs7685686 2089 TAAATTG * A * A * A * T * T * G * T * CXXXXXXXXX PS on each end and (A/G) TCATCA * A * T * C * mA Xbetween dN-mN and dN-dN WV- ACUAUTA 1084 mA * mCmUmAmU * T * A * A 1436XOOOXXXXX 5-15 hemimer 1 PS rs7685686 2090 ATAAATT* T * A * A * A * T * T * G * T XXXXXXXXX on each end and (A/G) GTCATC* C * A * T * C X between dN-mN and dN-dN WV- GACUUUU 1085rGrArCrUrUrUrUrUrCrUrGrGr 1437 OOOOOOOOO HTT rs7685686 HTT 2163 UCUGGUGUrGrArUrGrGrCrArArUrUrUrA OOOOOOOOO rs7685686 AUGGCAA rUrUrArArUrArGOOOOOOOOO UUUAUUA OOOO AUAG WV- GACUUUU 1086 rGrArCrUrUrUrUrUrCrUrGrGr1438 OOOOOOOOO HTT rs7685686 HTT 2164 UCUGGUG UrGrArUrGrArCrArArUrUrUrAOOOOOOOOO rs7685686 AUGACAA rUrUrArArUrArG OOOOOOOOO UUUAUUA OOOO AUAGWV- UAAAUTG 1087 mU * SmAmAmAmU * ST * 1439 SOOOSSSSSR 5-10-5 2′ OMe-HTT 2269 TCATCAC SG * ST * SC * SA * RT * SC * SSSSSOOOS DNA-2′-OMers7685686 CAGAAA SA * SC * SC * SmAmGmAmA Gapmer 1-3-11-3-1 * SmA(PS/PO) WV- AUAAATT 1088 mA * SmUmAmAmA * ST * ST 1440 SOOOSSSSSS5-10-5 2′ OMe- HTT 2270 GTCATCA * SG * ST * SC * SA * RT * SC RSSSSOOOSDNA-2′-OMe rs7685686 CCAGAA * SA * SC * SmCmAmGmA * Gapmer 1-3-11-3-1SmA (PS/PO) WV- AAUAAAT 1089 mA * SmAmUmAmA * SA * 1441 SOOOSSSSSS5-10-5 2′ OMe- HTT 2271 TGTCATC ST * ST * SG * ST * SC * SA * SRSSSOOOSDNA-2′-OMe rs7685686 ACCAGA RT * SC * SA * SmCmCmAmG Gapmer 1-3-11-3-1* SmA (PS/PO) WV- UAAUAAA 1090 mU * SmAmAmUmA * SA * 1442 SOOOSSSSSS5-10-5 2′ OMe- HTT 2272 TTGTCAT SA * ST * ST * SG * ST * SC * SSRSSOOOSDNA-2′-OMe rs7685686 CACCAG SA * RT * SC * SmAmCmCmA Gapmer 1-3-11-3-1* SmG (PS/PO) WV- AAUAAAT 1091 mA * SmAmUmAmA * SA * 1443 SOOOSSSSSSP10 stereopure HTT 2374 TGTCATC ST * ST * SG * ST * SC * SA * SRSSSSOOSanalogue of WV- rs7685686 ACCAGA RT * SC * SA * SC * 2071 5-11-4 2′-SmCmAmG * SmA OMe-DNA-2′-OMe Gapmer 1-3-12-2-1 (PS/PO) WV- UAAUAAA 1092mU * SmAmAmUmA * SA * 1444 SOOOSSSSSS P11 stereopure HTT 2375 TTGTCATSA * ST * ST * SG * ST * SC * SSRSSSOOS analogue of WV- rs7685686 CACCAGSA * RT * SC * SA * 20755-11-4 2′- SmCmCmA * SmG OMe-DNA-2′-OMeGapmer 1-3-12-2-1 (PS/PO) WV- GCACAAG 1093 mG * mCmAmCmA * A * G * 1445XOOOXXXXX P11 stereorandom HTT 2377 GGCACAGG * G * C * A * C * A * G * A * XXXXXXOOO analogue of WV- rs362307ACUUCC mCmUmUmC * mC X 932 5-10-5 2′- OMe-DNA-2′-OMe Gapmer and 1-3-11-3-1 (PS/PO) WV- GCACAAG 1094 mG * SmCmAmCmA * SA * 1446 SOOOSSSSSSP11 stereorandom HTT 2378 GGCACAG SG * SG * SG * SC * SA * SC *SRSSSOOOS analogue of WV- rs362307 ACUUCC RA * SG * SA * SmCmUmUmC932 5-10-5 2′- * SmC OMe-DNA-2′-OMe Gapmer and 1-3- 11-3-1 (PS/PO) WV-CACAAGG 1095 mC * mAmCmAmA * G * G * 1447 XOOOXXXXX P10 sereorandom HTT2379 GCACAGA G * C * A * C * A * G * A * C * XXXXXXOOO analogue of WV-rs362307 CUUCCA mUmUmCmC * mA X 933 5-10-5 2′- OMe-DNA-2′-OMeGapmer and 1-3- 11-3-1 (PS/PO) WV- CACAAGG 1096 mC * SmAmCmAmA * SG *1448 SOOOSSSSSS P10 stereopure HTT 2380 GCACAGASG * SG * SC * SA * SC * RA * RSSSSOOOS analogue of WV- rs362307 CUUCCASG * SA * SC * SmUmUmCmC 933 5-10-5 2′- * SmA OMe-DNA-2′-OMeGapmer and 1-3- 11-3-1 (PS/PO) WV- UAAAUTG 1097 mU * SmAmAmAmU * ST *1449 SOOOSSSSRS P8 5-10-5 2′ HTT 2416 TCATCACSG * ST * SC * RA * ST * SC * SSSSSOOOS OMe-DNA-2′-OMe rs7685686 CAGAAASA * SC * SC * SmAmGmAmA Gapmer 1-3-11-3-1 * SmA (PS/PO) WV- AUAAATT1098 mA * SmUmAmAmA * ST * ST 1450 SOOOSSSSSR P9 5-10-5 2′ HTT 2417GTCATCA * SG * ST * SC * RA * ST * SC SSSSSOOOS OMe-DNA-2′-OMe rs7685686CCAGAA * SA * SC * SmCmAmGmA * Gapmer 1-3-11-3-1 SmA (PS/PO) WV- AAUAAAT1099 mA * SmAmUmAmA * SA * 1451 SOOOSSSSSS P10 5-10-5 2′ HTT 2418TGTCATC ST * ST * SG * ST * SC * RA * RSSSSOOOS OMe-DNA-2′-OMe rs7685686ACCAGA ST * SC * SA * SmCmCmAmG Gapmer 1-3-11-3-1 * SmA (PS/PO) WV-UAAUAAA 1100 mU * SmAmAmUmA * SA * 1452 SOOOSSSSSS P11 5-10-5 2′ HTT2419 TTGTCAT SA * ST * ST * SG * ST * SC * SRSSSOOOS OMe-DNA-2′-OMers7685686 CACCAG RA * ST * SC * SmAmCmCmA Gapmer 1-3-11-3-1 * SmG(PS/PO) WV- UCCCCAC 1101 mU * SmCmCmCmC * SA * SC 1453 SOOOSSRSSSP6 5-10-5 (2′- HTT 2589 AGAGGGA * RA * SG * SA * SG * SG * SSSSSOOOSOMe-DNA-2′- rs2530595 GGAAGC SG * SA * SG * SmGmAmAmG OMe) 1-3-11-3-1(C/T) * SmC (PS/PO) Gapmer WV- CUCCCCA 1102 mC * SmUmCmCmC * SC * SA1454 SOOOSSSRSS P7 5-10-5 (2′- HTT 2590 CAGAGGG* SC * RA * SG * SA * SG * SG SSSSSOOOS OMe-DNA-2′- rs2530595 AGGAAG* SG * SA * SmGmGmAmA * OMe) 1-3-11-3-1 (C/T) SmG (PS/PO) Gapmer WV-CCUCCCC 1103 mC * SmCmUmCmC * SC * SC 1455 SOOOSSSSRS P8 5-10-5 (2′- HTT2591 ACAGAGG * SA * SC * RA * SG * SA * SG SSSSSOOOS OMe-DNA-2′-rs2530595 GAGGAA * SG * SG * SmAmGmGmA * OMe) 1-3-11-3-1 (C/T) SmA(PS/PO) Gapmer WV- UCCUCCC 1104 mU * SmCmCmUmC * SC * SC 1456 SOOOSSSSSRP9 5-10-5 (2′- HTT 2592 CACAGAG * SC * SA * SC * RA * SG * SA SSSSSOOOSOMe-DNA-2′- rs2530595 GGAGGA * SG * SG * SmGmAmGmG * OMe) 1-3-11-3-1(C/T) SmA (PS/PO) Gapmer WV- GUCCUCC 1105 mG * SmUmCmCmU * SC * SC 1457SOOOSSSSSS P10 5-10-5 (2′- HTT 2593 CCACAGA* SC * SC * SA * SC * RA * SG RSSSSOOOS OMe-DNA-2′- rs2530595 GGGAGG* SA * SG * SmGmGmAmG * OMe) 1-3-11-3-1 (C/T) SmG (PS/PO) Gapmer WV-GGUCCTC 1106 mG * SmGmUmCmC * ST * SC 1458 SOOOSSSSSS P11 5-10-5 (2′-HTT 2594 CCCACAG * SC * SC * SC * SA * SC * RA SRSSSOOOS OMe-DNA-2′-rs2530595 AGGGAG * SG * SA * SmGmGmGmA * OMe) 1-3-11-3-1 (C/T) SmG(PS/PO) Gapmer WV- GGGUCCT 1107 mG * SmGmGmUmC * SC * ST 1459 SOOOSSSSSSP12 5-10-5 (2′- HTT 2595 CCCCACA * SC * SC * SC * SC * SA * SC SSRSSOOOSOMe-DNA-2′- rs2530595 GAGGGA * RA * SG * SmAmGmGmG * OMe) 1-3-11-3-1(C/T) SmA (PS/PO) Gapmer WV- CGGGUCC 1108 mC * SmGmGmGmU * SC * SC 1460SOOOSSSSSS P13 5-10-5 (2′- HTT 2596 TCCCCAC* ST * SC * SC * SC * SC * SA SSSRSOOOS OMe-DNA-2′- rs2530595 AGAGGG* SC * RA * SmGmAmGmG * OMe) 1-3-11-3-1 (C/T) SmG (PS/PO) Gapmer WV-ACAGUAG 1109 mA * SmCmAmGmU * SA * 1461 SOOOSSRSSS P6 5-10-5 (2′- HTT2597 ATGAGGG SG * RA * ST * SG * SA * SG * SSSSSOOOS OMe-DNA-2′-(rs362331) AGCAGG SG * SG * SA * SmGmCmAmG OMe) 1-3-11-3-1 (C/T) * SmG(PS/PO) Gapmer WV- CACAGTA 1110 mC * SmAmCmAmG * ST * SA 1462 SOOOSSSRSSP7 5-10-5 (2′- HTT 2598 GATGAGG * SG * RA * ST * SG * SA * SG SSSSSOOOSOMe-DNA-2′- (rs362331) GAGCAG * SG * SG * SmAmGmCmA * OMe) 1-3-11-3-1(C/T) SmG (PS/PO) Gapmer WV- ACACAGT 1111 mA * SmCmAmCmA * SG * ST 1463SOOOSSSSRS P8 5-10-5 (2′- HTT 2599 AGATGAG * SA * SG * RA * ST * SG * SASSSSSOOOS OMe-DNA-2′- (rs362331) GGAGCA * SG * SG * SmGmAmGmC *OMe) 1-3-11-3-1 (C/T) SmA (PS/PO) Gapmer WV- CACACAG 1112mC * SmAmCmAmC * SA * SG 1464 SOOOSSSSSR P9 5-10-5 (2′- HTT 2600 TAGATGA* ST * SA * SG * RA * ST * SG SSSSSOOOS OMe-DNA-2′- (rs362331) GGGAGC* SA * SG * SmGmGmAmG * OMe) 1-3-11-3-1 (C/T) SmC (PS/PO) Gapmer WV-GCACACA 1113 mG * SmCmAmCmA * SC * SA 1465 SOOOSSSSSS P10 5-10-5 (2′-HTT 2601 GTAGATG * SG * ST * SA * SG * RA * ST RSSSSOOOS OMe-DNA-2′-(rs362331) AGGGAG * SG * SA * SmGmGmGmA * OMe) 1-3-11-3-1 (C/T) SmG(PS/PO) Gapmer WV- UGCACAC 1114 mU * SmGmCmAmC * SA * SC 1466 SOOOSSSSSSP11 5-10-5 (2′- HTT 2602 AGTAGAT * SA * SG * ST * SA * SG * RA SRSSSOOOSOMe-DNA-2′- (rs362331) GAGGGA * ST * SG * SmAmGmGmG * OMe) 1-3-11-3-1(C/T) SmA (PS/PO) Gapmer WV- GUGCACA 1115 mG * SmUmGmCmA * SC * 1467SOOOSSSSSS P12 5-10-5 (2′- HTT 2603 CAGTAGASA * SC * SA * SG * ST * SA * SSRSSOOOS OMe-DNA-2′- (rs362331) TGAGGGSG * RA * ST * SmGmAmGmG OMe) 1-3-11-3-1 (C/T) * SmG (PS/PO) Gapmer WV-AGUGCAC 1116 mA * SmGmUmGmC * SA * 1468 SOOOSSSSSS P13 5-10-5 (2′- HTT2604 ACAGTAG SC * SA * SC * SA * SG * ST * SSSRSOOOS OMe-DNA-2′-(rs362331) AUGAGG SA * SG * RA * OMe) 1-3-11-3-1 (C/T) SmUmGmAmG * SmG(PS/PO) Gapmer WV- UCCCCAC 1117 mU * mCmCmCmC * A * C * A 1469 XOOOXXXXXP6 5-10-5 (2′- HTT 2605 AGAGGGA * G * A * G * G * G * A * G * XXXXXXOOOOMe-DNA-2′- r2530595 GGAAGC mGmAmAmG * mC X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- CUCCCCA 1118 mC * mUmCmCmC * C * A * C 1470 XOOOXXXXXP7 5-10-5 (2′- HTT 2606 CAGAGGG * A * G * A * G * G * G * A * XXXXXXOOOOMe-DNA-2′- r2530595 AGGAAG mGmGmAmA * mG X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- CCUCCCC 1119 mC * mCmUmCmC * C * C * A 1471 XOOOXXXXXP8 5-10-5 (2′- HTT 2607 ACAGAGG * C * A * G * A * G * G * G * XXXXXXOOOOMe-DNA-2′- r2530595 GAGGAA mAmGmGmA * mA X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- UCCUCCC 1120 mU * mCmCmUmC * C * C * C 1472 XOOOXXXXXP9 5-10-5 (2′- HTT 2608 CACAGAG * A * C * A * G * A * G * G * XXXXXXOOOOMe-DNA-2′- r2530595 GGAGGA mGmAmGmG * mA X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- GUCCUCC 1121 mG * mUmCmCmU * C * C * C 1473 XOOOXXXXXP10 5-10-5 (2′- HTT 2609 CCACAGA * C * A * C * A * G * A * G * XXXXXXOOOOMe-DNA-2′- r2530595 GGGAGG mGmGmAmG * mG X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- GGUCCTC 1122 mG * mGmUmCmC * T * C * C 1474 XOOOXXXXXP11 5-10-5 (2′- HTT 2610 CCCACAG * C * C * A * C * A * G * A * XXXXXXOOOOMe-DNA-2′- r2530595 AGGGAG mGmGmGmA * mG X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- GGGUCCT 1123 mG * mGmGmUmC * C * T * C 1475 XOOOXXXXXP12 5-10-5 (2′- HTT 2611 CCCCACA * C * C * C * A * C * A * G * XXXXXXOOOOMe-DNA-2′- r2530595 GAGGGA mAmGmGmG * mA X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- CGGGUCC 1124 mC * mGmGmGmU * C * C * T 1476 XOOOXXXXXP13 5-10-5 (2′- HTT 2612 TCCCCAC * C * C * C * C * A * C * A * XXXXXXOOOOMe-DNA-2′- r2530595 AGAGGG mGmAmGmG * mG X OMe) 1-3-11-3-1 (C/T)(P/PO) Gapmer WV- ACAGUAG 1125 mA * mCmAmGmU * A * G * 1477 XOOOXXXXXP6 5-10-5 (2′- HTT 2613 ATGAGGG A * T * G * A * G * G * G * A *XXXXXXOOO OMe-DNA-2′- (r362331) AGCAGG mGmCmAmG * mG X OMe) 1-3-11-3-1(C/T) (P/PO) Gapmer WV- CACAGTA 1126 mC * mAmCmAmG * T * A * G 1478XOOOXXXXX P7 5-10-5 (2′- HTT 2614 GATGAGG * A * T * G * A * G * G * G *XXXXXXOOO OMe-DNA-2′- (r362331) GAGCAG mAmGmCmA * mG X OMe) 1-3-11-3-1(C/T) (P/PO) Gapmer WV- ACACAGT 1127 mA * mCmAmCmA * G * T * A 1479XOOOXXXXX P8 5-10-5 (2′- HTT 2615 AGATGAG * G * A * T * G * A * G * G *XXXXXXOOO OMe-DNA-2′- (r362331) GGAGCA mGmAmGmC * mA X OMe) 1-3-11-3-1(C/T) (P/PO) Gapmer WV- CACACAG 1128 mC * mAmCmAmC * A * G * T 1480XOOOXXXXX P9 5-10-5 (2′- HTT 2616 TAGATGA * A * G * A * T * G * A * G *XXXXXXOOO OMe-DNA-2′- (r362331) GGGAGC mGmGmAmG * mC X OMe) 1-3-11-3-1(C/T) (P/PO) Gapmer WV- GCACACA 1129 mG * mCmAmCmA * C * A * 1481XOOOXXXXX P10 5-10-5 (2′- HTT 2617 GTAGATGG * T * A * G * A * T * G * A * XXXXXXOOO OMe-DNA-2′- (r362331) AGGGAGmGmGmGmA * mG X OMe) 1-3-11-3-1 (C/T) (P/PO) Gapmer WV- UGCACAC 1130mU * mGmCmAmC * A * C * 1482 XOOOXXXXX P11 5-10-5 (2′- HTT 2618 AGTAGATA * G * T * A * G * A * T * G * XXXXXXOOO OMe-DNA-2′- (r362331) GAGGGAmAmGmGmG * mA X OMe) 1-3-11-3-1 (C/T) (P/PO) Gapmer WV- GUGCACA 1131mG * mUmGmCmA * C * A * 1483 XOOOXXXXX P12 5-10-5 (2′- HTT 2619 CAGTAGAC * A * G * T * A * G * A * T * XXXXXXOOO OMe-DNA-2′- (r362331) TGAGGGmGmAmGmG * mG X OMe) 1-3-11-3-1 (C/T) (P/PO) Gapmer WV- AGUGCAC 1132mA * mGmUmGmC * A * C * 1484 XOOOXXXXX P13 5-10-5 (2′- HTT 2620 ACAGTAGA * C * A * G * T * A * G * A * XXXXXXOOO OMe-DNA-2′- (r362331) AUGAGGmUmGmAmG * mG X OMe) 1-3-11-3-1 (C/T) (P/PO) Gapmer WV- GGCACAA 1133GGCACAAGGGCACAGACTTC 1485 OOOOOOOOO DNA version of HTT 2623 GGGCACAOOOOOOOOO WV-1092 rs362307 GACTTC O (C/T) WV- GGCACAA 1134mG * SmGmCmAmC * SA * 1486 SOOOSSSSSS WV-1092 analogue rs362307 2659GGGCACA SA * SG * SG * SG * SC * SA * SSSSSOOOS with All Sp Human GACUUCSC * SA * SG * SmAmCmUmU stereochemistry HTT * SmC WV- GGGUCCT 1135mG * SmG * SmGmUmC * SC 1487 SSOOSSSSSSS P12 5-10-5 (2′- HTT 2671CCCCACA * ST * SC * SC * SC * SC * SA SRSSOOSS OMe-DNA-2′- rs2530595GAGGGA * SC * RA * SG * SmAmGmG * OMe) 2-2-11-2-2 (C/T) SmG * SmA(PS/PO) Gapmer with Sp wings WV- GGGUCCT 1136 mG * RmG * RmGmUmC * SC1488 RROOSSSSSS P12 5-10-5 (2′- HTT 2672 CCCCACA* ST * SC * SC * SC * SC * SA SSRSSOORR OMe-DNA-2′- rs2530595 GAGGGA* SC * RA * SG * SmAmGmG * OMe) 4-11-4 (C/T) RmG * RmA (PS/PO) Gapmerwith Rp wings WV- GGGUCCT 1137 mG * SmG * SmG * SmU * 1489 SSSSSSSSSSSP12 5-10-5 (2′- HTT 2673 CCCCACA SmC * SC * ST * SC * SC * SC SRSSSSSSOMe-DNA-2′- rs2530595 GAGGGA * SC * SA * SC * RA * SG * OMe) 2-2-11-2-2(C/T) SmA * SmG * SmG * SmG * (PS/PO) Gapmer SmA with Sp wings WV-GGGUCCT 1138 mG * RmG * RmG * RmU * 1490 RRRRSSSSSSS P12 5-10-5 (2′- HTT2674 CCCCACA RmC * SC * ST * SC * SC * SC SRSSRRRR OMe-DNA-2′- rs2530595GAGGGA * SC * SA * SC * RA * SG * OMe) 2-2-11-2-2 (C/T)SmA * RmG * RmG * RmG * (PS/PO) Gapmer RmA with Rp wings WV- GGGUUCT1139 mG * SmGmGmUmU * SC * ST 1491 SOOOSSSSSS P12 analogue of HTT 2675CCCCACA * SC * SC * SC * SC * SA * SC SSRSSOOOS WV-2595 with G:Urs2530595 GAGGGA * RA * SG * SmAmGmGmG * mismatch at (C/T) SmAposition 5 WV- GGCACAA 1140 mG * RmGmCmAmC * SA * 1492 ROOOSSSSSSWV-1092 analogue rs362307 2676 GGGCACA SA * SG * SG * SG * SC * SA *SSSSSOOOS for CMC Human GACUUC SC * SA * SG * SmAmCmUmU HTT * SmC WV-GGCACAA 1141 mG * SmGmCmAmC * RA * 1493 SOOORSSSSS WV-1092 analoguers362307 2682 GGGCACA SA * SG * SG * SG * SC * SA * SSSSSOOOS for CMCHuman GACUUC SC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1142mG * SmGmCmAmC * SA * 1494 SOOOSRSSSS WV-1092 analogue rs362307 2683GGGCACA RA * SG * SG * SG * SC * SA * SSSSSOOOS for CMC HumanSC * SA * SG * SmAmCmUmU HTT GACUUC * SmC WV- GGCACAA 1143mG * SmGmCmAmC * SA * 1495 SOOOSSRSSS WV-1092 analogue rs362307 2684GGGCACA SA * RG * SG * SG * SC * SA * SSSSSOOOS for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1144mG * SmGmCmAmC * SA * 1496 SOOOSSSRSS WV-1092 analogue rs362307 2685GGGCACA SA * SG * RG * SG * SC * SA * SSSSSOOOS for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1145mG * SmGmCmAmC * SA * 1497 SOOOSSSSRS WV-1092 analogue rs362307 2686GGGCACA SA * SG * SG * RG * SC * SA * SSSSSOOOS for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1146mG * SmGmCmAmC * SA * 1498 SOOOSSSSSR WV-1092 analogue rs362307 2687GGGCACA SA * SG * SG * SG * RC * SA * SSSSSOOOS for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1147mG * SmGmCmAmC * SA * 1499 SOOOSSSSSS WV-1092 analogue rs362307 2688GGGCACA SA * SG * SG * SG * SC * RA * RSSSSOOOS for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1148mG * SmGmCmAmC * SA * 1500 SOOOSSSSSS WV-1092 analogue rs362307 2689GGGCACA SA * SG * SG * SG * SC * SA * SRSSSOOOS for CMC Human GACUUCRC * SA * SG * SmAmCmUmU HTT * SmC WV- GGCACAA 1149mG * SmGmCmAmC * SA * 1501 SOOOSSSSSS WV-1092 analogue rs362307 2690GGGCACA SA * SG * SG * SG * SC * SA * SSSRSOOOS for CMC Human GACUUCSC * SA * RG * SmAmCmUmU HTT * SmC WV- GGCACAA 1150mG * SmGmCmAmC * SA * 1502 SOOOSSSSSS WV-1092 analogue rs362307 2691GGGCACA SA * SG * SG * SG * SC * SA * SSSSROOOS for CMC Human GACUUCSC * SA * SG * RmAmCmUmU HTT * SmC WV- GGCACAA 1151mG * SmGmCmAmC * SA * 1503 SOOOSSSSSS WV-1092 analogue rs362307 2692GGGCACA SA * SG * SG * SG * SC * SA * SSSSSOOOR for CMC Human GACUUCSC * SA * SG * SmAmCmUmU HTT * RmC WV- GGCAC mG * SmGmCmAmC SOOOWV-1092 fragment rs362307 2728 for CMC Human HTT WV- GGCACmG * RmGmCmAmC ROOO WV-1092 fragment rs362307 2729 for CMC Human HTT WV-ACUUC mAmCmUmU * SmC OOOS WV-1092 fragment rs362307 2730 for CMC HumanHTT WV- ACUUC mAmCmUmU * RmC OOOR WV-1092 fragment rs362307 2731 for CMCHuman HTT WV- GGCACAA 1152 mG * SmGmCmAmC * SA * 1504 SOOOSSSSSSWV-1092 for CM rs362307 2732 GGGCACA SA * SG * SG * SG * SC * RA *RSRSSOOOS Human GACUUC SC * RA * SG * SmAmCmUmU HTT * SmC Abbreviations:2\′: 2′ 3\′: 3′ 5\′: 5′ 307: SNP rs362307 C6: C6 amino linker F, f: 2′-FHtt, HTT: Huntingtin gene or Huntington's Disease Laurie, Myristic,Palmitic, Stearic, Oleic, Linoleic, alpha-Linoleic, gamma-Linoleic, DHA,Turbinaric, Dilinoleic: Laurie acid, Myristic acid, Palmitic acid,Stearic acid, Oleic acid, Linoleic acid, alpha-Linoleic acid,gamma-Linoleic acid, docosahexaenoic acid, Turbinaric acid, Dilinoreicacid, respectively. muHtt or muHTT: mutant Huntingtin gene or geneproduct OMe: 2′-OMe O, PO: phoshodiester (phosphate) *, PS:Phosphorothioate R, Rp: Phosphorothioate in Rp conformation S, Sp:Phosphorothioate in Sp conformation WV: WV- WV-: WV X: Phosphorothioate,stereorandom

EQUIVALENTS

Having described some illustrative embodiments of the disclosure, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the disclosure. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the disclosure. The presentdisclosure is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of thedisclosure and other functionally equivalent embodiments are within thescope of the disclosure. Various modifications of the disclosure inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of thedisclosure are not necessarily encompassed by each embodiment of thedisclosure.

1-41. (canceled)
 42. A method for treating Huntington's disease in asubject, comprising administering to the subject a therapeuticallyeffective amount of an oligonucleotide having the structure of: mG *SmGmCmAmC * SA * SA * SG * SG * SG * SC * SA * SC * RA * SG *SmAmCmUmU * SmC (SEQ ID NO: 360), or a pharmaceutically acceptable saltthereof, wherein: S represents a Sp phosphorothioate; R represents a Rpphosphorothioate; and m represents a 2′-OMe modification to anucleoside.
 43. The method of claim 42, wherein the oligonucleotide isin a salt form.
 44. The method of claim 42, wherein the oligonucleotideis a sodium salt.
 45. The method of claim 42, wherein the subject has anrs362307 allele which is associated with Huntington's disease and is100% complementary to the base sequence of the oligonucleotide.
 46. Themethod of claim 43, wherein the subject has an rs362307 allele which isassociated with Huntington's disease and is 100% complementary to thebase sequence of the oligonucleotide.
 47. The method of claim 44,wherein the subject has an rs362307 allele which is associated withHuntington's disease and is 100% complementary to the base sequence ofthe oligonucleotide.
 48. A method for treating Huntington's disease in asubject, comprising administering to the subject a pharmaceuticalcomposition which comprises a therapeutically effective amount of anoligonucleotide and at least one pharmaceutically acceptable inactiveingredient selected from pharmaceutically acceptable diluents,pharmaceutically acceptable excipients, and pharmaceutically acceptablecarriers, wherein the oligonucleotide has the structure of: mG *SmGmCmAmC * SA * SA * SG * SG * SG * SC * SA * SC * RA * SG* SmAmCmUmU *SmC (SEQ ID NO: 360), or a pharmaceutically acceptable salt thereof,wherein: * S represents a Sp phosphorothioate; *R represents a Rpphosphorothioate; and m represents a 2′-OMe modification to anucleoside.
 49. The method of claim 48, wherein the oligonucleotide isin a salt form.
 50. The method of claim 48, wherein the oligonucleotideis a sodium salt.
 51. The method of claim 48, wherein the subject has anrs362307 allele which is associated with Huntington's disease and is100% complementary to the base sequence of the oligonucleotide.
 52. Themethod of claim 49, wherein the subject has an rs362307 allele which isassociated with Huntington's disease and is 100% complementary to thebase sequence of the oligonucleotide.
 53. The method of claim 50,wherein the subject has an rs362307 allele which is associated withHuntington's disease and is 100% complementary to the base sequence ofthe oligonucleotide.